Process for the preparation of DNA gyrase inhibitors

ABSTRACT

Bicyclic derivatives of the general formula ##STR1## wherein X 1  is --S-- or --SO--; 
     X 2  is --CO-- or --CS--; 
     R 1  is hydrogen, halogen or lower alkyl optionally substituted by halogen or lower alkoxy; 
     R 2  and R 3  are each independently hydrogen, lower alkyl, halogen, amino, lower alkylamino, di-lower alkylamino, acylamino, lower alkexy, lower alkoxymethoxy or a group OR 4  ; 
     R 4  is hydrogen or an easily hydrolyzable group; 
     R 5  is hydrogen, optionally esterified carboxy or amidated (thio)carboxy, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted acyl or heterocyclyl; 
     R 6  and R 7a  are each independently hydrogen or lower alkyl; 
     R 7b  is hydrogen, optionally substituted hydroxy, --NR--A or --N═B, in which R is hydrogen or lower alkyl, A is hydrogen, optionally substituted alkyl, lower cycloalkyl, iminoyl, (thio)acyl, esterified carboxy or amidated (thio)carboxy and B is lower alkylidene; 
     R 7a  and R 7b  together represent oxo, lower alkoxycarbonylmethylidene or optionally substituted hydroxyimino; and 
     R 8  is hydrogen, optionally substituted alkyl, optionally esterified carboxy or amidated (thio)carboxy; provided that no more than two of R 1  -R 3  are nitrogen-containing groups; no more than two of R 1  -R 3  are oxygen containing groups and no more than two of R 1  -R 3  are either nitrogen containing or oxygen containing groups; 
     and pharmaceutically acceptable salts of the compounds of formula I carrying an acidic and/or basic substituent. 
     The products are antimicrobially active.

This is a division of application Ser. No. 08/177,483, filed Jan. 6,1994, now U.S. Pat. No. 5,399,741 which is a division of applicationSer. No. 07/952,537, filed Dec. 9, 1992, now U.S. Pat. No. 5,294,609,filed as International Application PCT/EP92/00809, filed on Apr. 9, 1992and which designated in the U.S.

The invention relates to novel DNA gyrase inhibitors, which are bicyclicderivatives of the general formula ##STR2## wherein X¹ is --S-- or--SO--;

X² is --CO-- or --CS--;

R¹ is hydrogen, halogen or lower alkyl optionally substituted by halogenor lower alkoxy;

R² and R³ are each independently hydrogen, lower alkyl, halogen, amino,lower alkylamino, di-lower alkylamino, acylamino, lower alkoxy, loweralkoxymethoxy or a group OR⁴ ;

R⁴ is hydrogen or an easily hydrolyzable group;

R⁵ is hydrogen, optionally esterified carboxy or amidated (thio)carboxy,optionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted acyl or heterocyclyl;

R⁶ and R^(7a) are each independently hydrogen or lower alkyl;

R^(7b) is hydrogen, optionally substituted hydroxy, --NR--A or --N═B, inwhich R is hydrogen or lower alkyl, A is hydrogen, optionallysubstituted alkyl, lower cycloalkyl, iminoyl, (thio)acyl, esterifiedcarboxy or amidated (thio)carboxy and B is lower alkylidene;

R^(7a) and R^(7b) together represent oxo, loweralkoxycarbonylmethylidene or optionally substituted hydroxyimino; and

R⁸ is hydrogen, optionally substituted alkyl, optionally esterifiedcarboxy or amidated (thio)carboxy; provided that no more than two of R¹-R³ are nitrogen-containing groups; no more than two of R¹ -R³ areoxygen containing groups and no more than two of R¹ -R³ are eithernitrogen containing or oxygen containing groups;

and pharmaceutically acceptable salts of the compounds of formula Icarrying an acidic and/or basic substituent.

Hereinabove and in the following, reference to the word "lower" such asin "lower alkyl", "lower alkoxy", "lower alkanoyl" etc. refers tohydrocarbon groups containing up to and including 6, preferably 1-2,carbon atoms unless otherwise specified. Thus, e.g. "lower alkyl" in thefollowing, alone or in combination with other groups such as in "loweralkylamino", "di-lower alkylamino", "aryl-lower alkyl" etc. is e.g.methyl, ethyl, t-butyl, n-pentyl etc.; "lower alkoxy" has analogousmeanings; "lower alkenyl" alone or in combination with other groups suchas "lower cycloalkyl-lower alkenyl", "heterocyclyl-lower alkenyl" etc.is e.g. vinyl, 1- or 2-propenyl; "lower cycloalkyl" alone or incombination with other groups such as "lower cycloalkyl-lower alkenyl","lower cycloalkyl-lower alkyl" etc. is e.g. cyclopropyl, cyclobutyl,cyclohexyl; "lower alkynyl" alone or in combination with other groupssuch as "lower cycloalkyl-lower alkynyl" is e.g. ethynyl, 1- or2-propynyl; "lower alkanoyl" alone or in combination with other groupssuch as "lower alkanoyloxy" etc. is e.g. formyl, acetyl, propionyl,isobutyryl, pivaloyl etc.

Groups not specified by the word "lower", such as "alkyl", "alkoxy","alkenyl", "acyl" and "alkanoyl", are intended to refer to groupscontaining up to and including 14 carbon atoms unless otherwisespecified.

"(Thio)acyl" refers to an acyl group or a thioacyl group.

"Acyl" alone or in combination with other groups such as in "acylamino",is preferably derived from a carboxylic acid and is thus e.g. loweralkanoyl, e.g. formyl, acetyl, propionyl, isobutyryl, pivaloyl; loweralkenoyl, e.g. crotonoyl, isocrotonoyl; lower cycloalkanoyl, e.g.cyclopropylcarbonyl; aroyl, e.g. benzoyl, o-carboxy-benzoyl, p-toluoyl,p-anisoyl, naphthoyl; heterocyclylcarbonyl, e.g. furoyl, thenoyl."Thioacyl" has analogous meanings.

"Halogen" alone or in combination with other groups such as in"halogen-lower alkyl" etc. refers to all four halogens, i.e. chlorine,bromine, iodine, fluorine, unless otherwise indicated.

The expressions "lower alkenylalkyl" and "lower alkynylalkyl" areemployed to indicate that the double and triple bonds of these groupsare not connected with the first carbon atom (such as in vinyl, ethynyland 1-propynyl), but that these groups are limited to the less reactivegroups having their unsaturation in 2-, 3- and further positions. It isunderstood that "lower alkenylalkyl" and "lower alkynylalkyl" refer togroups containing up to and including 5 carbon atoms, e.g. 2-propenyl,2-butenyl, 3-butenyl, 2-methyl-2-propenyl, 2-propynyl, 2-butynyl,3-butynyl, 2-methyl-2-propynyl.

Easily hydrolyzable groups --OR⁴ are e.g. lower alkanoyloxy groups suchas formyloxy, acetoxy, propionyloxy, isobutyryloxy, pivaloyloxy or loweralkoxycarbonyloxy groups such as methoxycarbonyloxy orethoxycarbonyloxy.

The above definition of R¹ -R³ signifies that no more than two of thesesubstituents are nitrogen-containing groups, i.e. amino, loweralkylamino, di-lower alkylamino and/or acylamino; moreover, no more thantwo of the substituents R¹ -R³ are oxygen containing groups, i.e. loweralkoxy-lower alkyl (R¹) and acylamino, lower alkoxy, lower alkoxymethoxyand/or a group OR⁴ (R², R³); moreover, R¹ -R³ can together only containa maximum of two of said nitrogen and/or oxygen containing groups.

R⁵ is hydrogen, optionally esterified carboxy or amidated (thio)carboxy,optionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted acyl or heterocyclyl. Exemplary of esterified carboxy andamidated (thio)carboxy groups R⁵ are groups of the general formula

    --COOY, --CONHR.sup.10, --CSNHR.sup.10, --CON(OH)R.sup.10, --CSN(OH)R.sup.10, --CONR.sup.10 Y, --CSNR.sup.10 Y

wherein R¹⁰ is hydrogen or alkyl and Y is alkyl, alkenylalkyl,alkynylalkyl, lower cycloalkyl, lower cycloalkyl-lower alkyl, lowercycloalkyl-lower alkenylalkyl, heterocyclyl, heterocyclyl-lower alkyl,heterocyclyl-lower alkenylalkyl, aryl, aryl-lower alkyl or aryl-loweralkenylalkyl or wherein the residue --NR¹⁰ Y represents the aminoresidue of an α-amino acid or peptide or a 5 to 7 membered saturatedN-heterocyclus optionally containing a further N or an O or S atom.

Thus, possible meanings for Y are alkyl, e.g. methyl, ethyl, isopropyl,t-butyl, n-pentyl, n-decyl, etc., alkenylalkyl, e.g. 2-propenyl;alkynylalkyl, e.g. 2-propynyl, 3-butynyl; lower cycloalkyl, e.g.cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl; lower cycloalkyl-loweralkyl, e.g. cyclopropylmethyl, cyclopropylethyl; lower cycloalkyl-loweralkenylalkyl, e.g. cyclopropyl-2-propenyl; heterocyclyl, such as a 5 to7 membered saturated or unsaturated heterocycle containing 1-4 nitrogenatoms and/or a sulfur or oxygen atom, e.g. 3-pyridyl, 2-pyrrolidy],2-pyrrolyl, 1-methyl-4-piperidyl, 1-ethoxycarbonyl-4-piperidinyl,thienyl, 4-thiazolyl; heterocyclyl-lower alkyl (where the heterocyclylmoiety is as defined above), e.g. furfuryl, thenyl, 4-thiazolyl-methyl,3-methyl-5-isoxazolyl-ethyl, 1-morpholinyl-methyl,4-methyl-1-piperazinylmethyl, 1-pyridinium-methyl; heterocyclyl-loweralkenylalkyl (where the heterocyclyl moiety is as defined above), e.g.2-pyrrolyl-2-propenyl, 2-thienyl-2-propenyl; aryl, e.g. phenyl, p-tolyl,o,m-dihydroxyphenyl, m,p-dihydroxyphenyl, p-methoxyphenyl (anisyl),m-methoxyphenyl, o,m-dimethoxyphenyl, 3,4,5-trimethoxyphenyl,p-trifiuoromethyl-phenyl, naphthyl; aryl-lower alkyl, e.g. benzyl,phenethyl; or aryl-lower alkenylalkyl, e.g. phenyl-2-propenyl. The saidheterocyclyl or aryl groups may be bound to a fused saturated orunsaturated ring which may contain 1-4 nitrogen atoms and/or a sulfur oroxygen atom to form e.g. a quinolinyl, quinoxalinyl, benzoxazolyl,benzthiazolyl, benzimidazolyl, indolyl, s-triazolo[1,5-a]pyrimidyl orpyrazolo[1,5-a]pyrimidinyl group.

The residue --NR¹⁰ Y can represent the amino residue of an α-amino acidor peptide. Such amino residues are e.g. ##STR3## The residue --NR¹⁰ Ycan also represent a 5 to 7 membered saturated N-heterocyclus optionallycontaining a further N or an O or S atom, e.g. pyrrolidyl, piperidino,morpholino, thiomorpholino.

The above group Y can be further substituted, e.g.

by halogen, i.e. fluorine, chlorine, bromine or iodine;

by amino (such as in 2-amino-4-thiazolyl);

by lower alkylamino, e.g. methylamino;

by di-lower-alkylamino, e.g. dimethylamino;

by a quaternary ammonium group such as tri-lower alkylammonium, e.g.trimethylammonium, 1-pyridinium, 1-lower-alkyl-morpholinium, e.g.1-methyl-morpholinium, or 1-quinuclidinium (in such case the positivecharge of the quaternary ammonium group is neutralized by apharmaceutically acceptable anion such as those exemplified below underthe acid addition salts of the compounds of formula I. The anion canalso be the deprotonated moiety of a carboxy group present in thecompound of formula I, in which the compound is present in the form of azwitterion);

by acylamino, e.g. acetamido, benzamido, p-toluoylamido;

by amidino (optionally mono-, di- or tri-substituted by lower alkyl,viz. a group of the formula --C(NRR')═NR" where R, R' and R" arehydrogen or lower alkyl);

by iminoyl (optionally mono or disubstituted by lower alkyl, viz. agroup of the formula --CR═NR', where R and R' are hydrogen or loweralkyl);

by hydroxy;

by lower alkoxy, e.g. methoxy, ethoxy;

by lower alkylthio, e.g. methylthio, ethylthio;

by lower cycloalkoxy, e.g. cyclopropoxy;

by lower cycloalkylthio, e.g. cyclopropylthio;

by lower alkenylalkoxy, e.g. 2-propenoxy;

by lower alkenylalkylthio, e.g. 2-propenylthio;

by aryloxy, e.g. phenoxy, p-telyloxy, naphthyloxy;

by arylthio. e.g. phenylthio, p-tolylthio, naphthylthio;

by heterocyclyloxy, e.g. furyloxy or thienyloxy;

by heterocyclylthio, e.g. furylthio, thiienylthio or(2,5-dihydro-6-hydroxy-2-methyl-5-oxo-as-triazin-3-yl)thio;

by acyloxy, the acyl moiety of which is preferably derived from acarboxylic acid and is thus e.g. lower alkanoyl, e.g. formyl, acetyl,propionyl, isobutyryl, pivaloyl; lower alkenoyl, e.g. crotonoyl,isocrotonoyl; lower cycloalkanoyl, e.g. cyclopropylcarbonyl: aroyl, e.g.benzoyl, p-chlorbenzoyl, p-toluoyl, p-anisoyl, naphthoyl;heterocyclylcarbonyl, e.g. furoyl, thenoyl;

by lower alkylsulfinyl or -sulfonyl, e.g. methylsulfinyl or-sulfonyl orethylsulfinyl or-sulfonyl;

by lower alkenylalkylsulfinyl or -sulfonyl, e.g. 2-propenylsulfinyl or-sulfonyl;

by lower cycloalkylsulfinyl or -sulfonyl, e.g. cyclopropylsulfinyl or-sulfonyl;

by arylsulfinyl or -sulfonyl, e.g. phenylsulfinyl or-sulfonyl orp-tolylsulfinyl or -sulfonyl;

by heterocyclylsulfinyl or-sulfonyl, e.g. furylsulfinyl or-sulfonyl orthienylsulfinyl or -sulfonyl;

by hydroxyirnino or lower alkoxyimino, e.g. methoxyimino.

The above groups Y can further be substituted by carboxy which isoptionally esterified or amidated, e.g. forming lower alkoxycarbonyl,carbamoyl or N-hydroxycarbamoyl (of which the last two may beN-substituted by lower alkyl or aryl).

Moreover, the above groups Y can be substituted by lower alkyl, e.g.methyl, ethyl, isopropyl; by halo-lower alkyl, e.g. 2-fluoroalkyl,trifiuoromethyl, trichloromethyl; by hydroxy-lower alkyl, e.g.hydroxymethyl, 2-hydroxyethyl; by lower alkoxy-lower alkyl, e.g.methoxymethyl; ethoxymethyl; by lower cydoalkyl e.g. cyclopropyl,cyclobutyl, cydohexyl; by lower alkenyl, e.g. vinyl, 2-propenyl; byaryl, e.g. phenyl, p-telyl, p-methoxyphenyl, naphthyl; by heterocydyl,e.g. 2-pyrrolidyl, 2-pyrrolyl, 2-thienyl; by oxo, cyano, nitro, azido orsulfamoyl which may be substituted by lower alkyl or aryl, e.g.methylsulfamoyl, dimethylsulfamoyl, phenylsulfamoyl.

Examples of unsubstituted alkyl, alkenyl or acyl groups R⁵ are givenabove for these expressions. These groups can, however, also besubstituted, e.g. by hydroxy, halogen, optionally lower alkyl- oraryl-substituted carbamoyloxy, carboxy, N-hydroxycarbamoyloxy or a groupof the general formula

in which Y has the meaning given above, Q is --O--, --S--, --SO₂ --,--COO--, --OCO--, --CONR¹⁰ --, --NR¹⁰ --, --NR¹⁰ --CO--, --NR¹⁰ SO₂ --,--NR¹⁰ COO-- or --NR¹⁰ CONR¹⁰ --, R¹⁰ has the above meaning and m iszero or 1.

Consequently, substituted alkyl, alkenyl and acyl groups include groupssuch as

hydroxymethyl

bromomethyl

fiuoroethyl

aminomethyl

2-carboxyethyl

4-fluoro-1-butenyl

(carbamoyloxy)methyl

[(phenylcarbamoyl)oxy]methyl

methoxymethyl

[(4-carbamoylphenyl)thio]methyl

(ethoxycarbonyl)acetyl

2-[(2-thiazolyl)carbamoyl]ethyl

(dimethylamino)methyl

cyclopentanecarboxamidomethyl

(4-aminobenzenesulfonamido)methyl

[1-(benzyloxy)fomamido]methyl

(3-phenyl ureido)methyl.

The above enumerated definition of Y including its further substitutionpossibilities is to be understood pragmatically such that apparentlymeaningless combinations such as "alkyl substituted alkyl", "alkenylsubstituted alkenyl", "alkyl substituted alkenyl" etc. are intended tomeans the abbreviated groups, i.e., the just stated expressions mean"alkyl", "alkenyl", and "alkenyl", respectively.

R⁵ can also refer to a heterocyclyl group, in particular to a 5 memberedheterocycle containing 1-4 nitrogen atoms and/or a sulfur or oxygenatom, optionally further substituted by lower alkyl, such as methyl, oramino.

R^(7b) is hydrogen, optionally substituted hydroxy, --NR--A or --N═B, inwhich R is hydrogen or lower alkyl, A is hydrogen, optionallysubstituted alkyl, lower cycloalkyl, iminoyl, (thio)acyl, esterifiedcarboxy or amidated (thio)carboxy and B is lower alkylidene. When R^(7b)is substituted hydroxy the substituted is preferable selected among acyland lower alkyl groups as defined above and aryl, preferably phenyl,which may be substituted by one or two nitro groups. A in its meaning asoptionally substituted alkyl or lower cycloalkyl have a significance asexplained above for R⁵. A in its meaning as iminoyl, (thio)acyl,esterified carboxy or amidated (thio)carboxy can be groups of theformula ##STR4## wherein R is hydrogen or lower alkyl, n is one or two,Y¹ is hydrogen or the group Y, Y² is hydrogen, lower alkenyl, loweralkynyl or the group Y and X² and Y are as above.

A in its meaning as an acyl group --(X²)_(n) --Y² can be the acylresidue of an α-amino acid or peptide or an N-protected derivativethereof. Such acyl residues are e.g. ##STR5## wherein M is hydrogen,lower alkanoyl, such as acetyl, or lower alkoxycarbonyl, such asmethoxycarbonyl, ethoxycarbonyl or t-butoxycarbonyl ("BOC"),

and others. Two such residues can be connected with the formation of adipeptide group, e.g. ##STR6## wherein M is as above.

Lower alkylidene groups B are e.g. groups of the general formulas

    ═CHY                                                   (e)

    ═C(Y).sub.2                                            (f)

    ═CR.sup.10 --NR.sup.10, Y.sup.1                        (g)

wherein R¹⁰, Y and Y¹ are as above.

R⁸ in its meaning a optionally substituted alkyl, lower cycloalkyl,optionally esterified carboxy or amidated (thio)carboxy have asignificance as explained above for R⁵.

Preferred meanings for various substituents are:

X¹ : --S--;

R¹, R³, R⁶, R^(7a), R⁸ : Hydrogen or methyl;

R² : Lower alkoxy, particularly methoxy or a group --OR⁴, particularlyhydroxy;

R⁴ : Hydrogen;

R⁵ : C₁ -C₅ Alkoxcycarbonyl, allylamido, prop-2-ynylamido,4-methyl-thiazol-2-yl, 3-methyl-1,2,4-oxadiazol-5-yl;

R^(7b) : Methoxycarbonylamino, hydroxy, formamido,t-butoxycarbonylamino, hydrogen, (thio)acetamido.

Preferred compounds of the invention are:

Methyl (4R,7S)-1,3,4,5,6,7,8,10-octahydro-14-hydroxy-12-methoxy-4-methoxycarbonyl-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamate,

methyl(4R,7S)-1,3,4,5,6,7,8,10-octahydro-7,14-dihydroxy-12-methoxy-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylate,

ethyl(4R,7S)-7-formamido-1,3,4,5,6,7,8,10-octahydro-14-hydroxy-12-methoxy-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylate,

(4R, 7 S)-7-t-butoxycarbonylamino-1,3,4,5,6,7,8,10-octahydro-14-hydroxy-12-methoxy-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylicacid allylamide,

(4R,7S)-7-t-butoxycarbonylamino-1,3,4,5,6,7,8,10-octahydro-14-hydroxy-12-methoxy-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylicacid prop-2-ynylamide,

methyl(4R)-1,3,4,5,6,7,8,10-octahydro-14-hydroxy-12-methoxy-10-oxo-6-thioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylate,

ethyl(4R,7S)-1,3,4,5,6,7,8,10-octahydro-14-hydroxy-12-methoxy-11-methyl-6,10-dioxo-7-(thioacetamido)-9,2,5-benzoxathiaazacyclododecine-4-carboxylate,

methyl(4R,7S)-7-(acetamido)-1,3,4,5,6,7,8,10-octahydro-14-hydroxy-12-methoxy-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylate,

t-butyl(4R,7S)-4,5,6,7,8-tetrahydro-12,14-dihydroxy-11-methyl-4-(4-methyl-thiazol-2-yl)-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamate,

t-butyl(4R,7S)-4,5,6,7,8-tetrahydro-14-hydroxy-12-methoxy-11-methyl-4(4-methyl-thiazol-2-yl)-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamate,

(4R,7S)-4,5,6,7,8-tetrahydro-7,12,14-trihydroxy-11-methyl-4-(3-methyl-1,2,4-oxadiazol-5-yl)-9,2,5-benzoxathiaazacyclododecine-6,10-dione,

(4R,7S)-4,5,6,7,8-tetrahydro-7,14-dihydroxy-12-methoxy-11-methyl-4-(3-methyl-1,2,4-oxadiazol-5-yl)-9,2,5-benzoxathiaazacyclododecine-6,10-dione,

N-[[(4R,7S)-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-7-[[(3R)-3-hydroxy-1-L-seryl-L-propyl]amino]-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecin-4-yl]carbonyl]-L-alanine

and pharmaceutically acceptable salts of any one of these compoundscarrying an acidic and/or basic substituent.

Other, exemplary compounds of the invention are:

t-Butyl(4R,7S)-12,14-diacetoxy-1,3,4,5,6,7,8,10-octahydro-4-methoxycarbonyl-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamate,

t-butyl(4R,7S)-1,3,4,5,6,7,8,10-octahydro-12,14-hydroxy-4-methoxycarbonyl-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamate,

(4R,7S)-7-(1-t-butoxyformamido)-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylicacid,

(4R,7S)-7-amino-1,3,4,5,6,7,8,10-octahydro-12,14-dihydro-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazabicyclododecine-4-carboxylicacid,

methyl(4R,7S)-12,14-diacetoxy-7-amino-1,3,4,5,6,7,8,10-octahydro-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylate,

methyl(4R,7S)-7-amino-1,3,4,5,6,7,8,10-octahydro-12,14-dihydro-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylate,

t-butyl(S)-12,14-diacetoxy-1,3,4,5.6,7,8,10-octahydro-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7carbamate,

t-butyl(S)-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamate,

(S)-7-amino-4,5,7,8-tetrahydro-12,14-dihydroxy-11-methyl-9,2,5-benzoxathiaazacyclododecine-6,10(1H,3H)-dione,

methyl(4R,7S)-12,14-diacetoxy-7-[(Z)-2-(2-amino-4-thiazolyl)-2-(methoxyimino)acetamido]-1,3,4,5,6,7,8,10-octahydro-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylate,

methyl(4R,75)-7-[(Z)-2-(2-amino-4-thiazolyl)-2-(methoxyimino)acetamido]-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylate

and pharmaceutically acceptable salts of any one of these compoundscarrying an acidic and/or basic substituent.

Compounds of formula I carrying an acidic, e.g. carboxylic, substituentform pharmaceutically acceptable salts with bases. Examples of salts ofcompounds of formula I are the alkali metal salts, for examples thesodium and potassium salts, the ammonium salts, the alkaline earth metalsalts, for example calcium salts, the salts with organic bases, forexample with amines such as diisopropylamine, benzylamine,dibenzylamine, triethanolamine, triethylamine,N,N-dibenzylethylenediamine, N-methylmorpholine, pyridine, piperazine,N-ethylpiperidine, N-methyl-D-glucamine and procaine or with amino acidssuch as arginine and lysine. Mono-, di-, tri-salts etc. can resultdepending on the number of acidic groups in the compounds of formula I.

Compounds of formula I which have a basic, e.g. amino, substituent alsoform acid addition salts with organic and inorganic acids. Examples ofacid addition salts of compounds of formula I are salts with mineralacids, for example hydrohalic acids such as hydrochloric acid, hydrogenbromide and hydrogen iodide, sulphuric acid, nitric acid, phosphoricacid and the like, salts with organic sulphonic acids, for example withalkyl- and arylsulphonic acids such as ethanesulphonic acid,p-toluenesulphonic acid, benzenesulphonic acid and the like, as well assalts with organic carboxylic acids, for example with acetic acid,tartaric acid, maleic acid, citric acid; benzoic acid, salicylic acid,ascorbic acid and the like.

The compounds of formula I and their pharmaceutically acceptable saltscan be manufactured in accordance with the invention by a process whichcomprises

a) cyclizing a carboxylic acid of the general formula ##STR7## in whichP is hydroxy and T is the group --X² --CR^(7a) R^(7b--CHR) ⁸ --OH or Tis hydrogen and P is the group --O--CHR⁸ --CR^(7a) R^(7b) --X² --OH andX¹, X² and R¹ -R⁸ are as above,

or a reactive derivative thereof, or

b) for the manufacture of a compound of formula I in which at least oneof R², R³ and R^(7b) is amino, reducing the nitro group(s) to amino in acompound of the general formula ##STR8## in which X¹, X², R¹, R⁴, R⁵,R⁶, R^(7a) and R⁸ are as above and R²⁰, R³⁰ and R^(70b) are as R², R³and R^(7b) above except that at least one of these substituentsrepresents nitro,

or

c) for the manufacture of a compound of formula I in which at least oneof R², R³ and R^(7b) represents acylamino, acylating the amino group(s)in a compound of the general formula ##STR9## in which X¹, X², R¹, R⁴,R⁵, R⁶, R^(7a) and R⁸ are as above and R²¹, R³¹ and R^(71b) and as R²,R³ and R^(7b) above except that at least one of these substituentsrepresents amino,

or

d) for the manufacture of a compound of formula I in which R^(7b) is thegroup --N═B reacting a compound of the general formula ##STR10## inwhich X¹, X², R¹ -R⁶, R^(7a) and R⁸ are as above, with a compound of thegeneral formula

    B═O

in which B is lower alkylidene,

or

e) for the manufacture of a compound of formula I in which R⁵ isesterified carboxy or amidated carboxy, reacting a compound of thegeneral formula ##STR11## in which X¹, X², R¹ -R⁴ and R⁶ -R⁸ are asabove and X³ is oxygen or sulfur,

with an agent yielding the corresponding ester or amide moiety, or

f) for the manufacture of a compound of formula I, in which any of R²,R³, R⁵ and/or R^(7b) is or contains an amino, hydroxy and/or carboxygroup cleaving off (a) protection group(s) in a compound of the generalformula ##STR12## in which X¹, X², R¹, R⁴, R⁶, R^(7a) and R⁸ are asabove and R²², R³², R⁵² and R^(72b) are as R², R³, R⁵ and R^(7b) exceptthat any amino, hydroxy and/or carboxy group is protected,

or

g) for the manufacture of a compound of formula I in which X¹ is--SO-oxidizing a compound of formula I in which X¹ is --S--, or

h) for the manufacture of a pharmaceutically acceptable salt of acompound of formula I carrying an acidic and/or basic substituentconverting such compound of formula I into such salt.

The cyclization in accordance with variant a) of the process inaccordance with the invention consists of two types. One is anintramolecular esterification (lactonization) and utilizes startingmaterials of formula II in which P is hydroxy, viz. compounds of thegeneral formula ##STR13## in which X¹, X² and R¹ -R⁸ are as above, orreactive derivatives thereof. Preferred starting-materials are thereactive derivatives, viz. compounds corresponding to formula IIa, inwhich the carboxy function has been converted into a reactivederivative, preferably into a reactive derivative with anN-heteroaromatic thiol, in particular 2-mercaptopyridine or a di-loweralkyl substituted 2-mercaptoimidazole such as4-t-butyl-N-isopropyl-2-mercaptoimidazole.

These N-heteroaromatic thiol esters can be prepared by reacting thedisulfide corresponding to said thiol and triphenyl phosphine with thecarboxylic acid of formula IIa. The reaction proceeds in an aproticorganic solvent such as benzene, toluene, xylene or methylene chlorideand at a temperature between above -20° C. and +40° C., preferably atabout 0° C. and +20° C. The reaction can proceed already under the saidconditions. However, in general conversion is achieved to completion byheating the reaction mixture, preferably at reflux for about 0.1-20hours.

Instead of the above N-heteroaromatic thiol ester an ester with a1-lower alkyl-2-halopyridinium salt, preferably1-methyl-2-chloropyridinium iodide, can be employed. For example, thestarting compound of formula IIa is reacted with e.g.1-methyl-2-chloropyridinium iodide in the presence of a tertiary aminesuch as triethylamine in an aprotic organic solvent such as acetonitrileor methylene chloride at a temperature between room temperature and theboiling point of the reaction mixture, preferably the latter.

According to a further alternative a reactive derivative with a di-loweralkyl azodicarboxylate, e.g. diethyl azodicarboxylate, and withtriphenylphosphine is employed. For example, diethyl azodicarboxylate isadded, dissolved in an aprotic organic solvent, such as benzene ortoluene, to a solution of the starting compound of formula IIa andtriphenylphosphine in the same solvent. The reaction can be carried outat a temperature between about -10° C. and +80° C., preferably at about0° C. to about +30° C.

According to a further alternative the starting compound of formula IIais cyclized with the aid of a mixture of a carbodiimide (such asdicyclohexylcarbodiimide), 4-(dimethylamino)pyridine and an acidaddition salt, e.g. the hydrochloride, of the latter. This reactionpreferably proceeds in an inert, aprotic organic solvent such astetrahydrofuran or, preferably, chloroform at a temperature betweenabout room temperature and reflux temperature, preferably at the latter.

The second type of the cyclization in accordance with variant a) of theprocess in accordance with the invention is an intramolecular amidationand utilizes starting materials of formula II in which T is hydrogen,viz. compounds of the general formula ##STR14## in which X¹, X² and R¹-R⁸ are as above, or reactive derivatives thereof. Reactive derivativesare compounds corresponding to formula IIb in which the carboxy functionhas been converted into a reactive derivative, preferably into an acidhalide, particularly the chloride: into a mixed acid anhydride,particularly with trifiuoroacetic acid or p-toluenesulfonic acid, orinto a reactive thiol ester, particularly a 2-pyridine thiol ester.These derivatives are obtained in a manner known per se by reacting thestarting compound of formula IIb with the corresponding add or with thedisulfide corresponding to 2-pyridine thiol and triphenyl phosphine inthe above mentioned manner. The cyclization of the reactive derivativesof the carboxylic add of formula IIb proceeds in an aprotic solvent suchas toluene or methylene chloride at a temperature between about -20° C.and 130° C., preferably in the range of about 0° C. and 110° C.

The starting compounds of formula IIb themselves can be cyclized in thepresence of carboxylic acid activators such as 1-loweralkyl-2-halopyridinium salts, e.g. 1-methyl-2-chloropyridinium iodide,dicyclohexylcarbodiimide or N-ethyl-5-phenyl-isoxazolium-3'-sulfonate,preferably in the presence of an organic base such as triethylamine orN-methyl-morpholine. The reaction is carried out in an aprotic organicsolvent such as methylene chloride or acetonitrile and at a temperaturebetween about 0° C. and the boiling point of the reaction mixture.

The reduction of nitro groups R²⁰, R³⁰ and/or R^(70b) to amino instarting compounds of formula IIIa according to variant b) of theprocess in accordance with the invention can be carried out in a mannerknown per se, e.g. by reaction with zinc, iron or tin in the presence ofa mineral acid such as aqueous hydrochloric acid. The reaction ispreferably carried out at a temperature in the range of about 0° C. to50° C., optionally in the presence of a co-solvent such astetrahydrofuran.

The acylation of amino groups R²¹, R³¹ and/or R^(71b) in startingcompounds of formula IIIb according to variant c) of the process inaccordance with the invention can be carded out in a manner known perse, e.g. by reacting an amine of formula IIIb with the correspondingcarboxylic acid in the presence of a coupling agent such as acarbodiimlde, e.g. dicydohexylcarbodiimide, or a 1-loweralkyl-2-halopyridinium salt, e.g. 1-methyl-2-chloropyridinium iodide, inan inert solvent such as acetonitrile, dioxane or tetrahydrofuran. It isalso possible to use a reactive derivative of the said carboxylic acidsuch as e.g. an acid chloride, a mixed anhydride with another organicacid, e.g. trifiuoroacetic acid or benzene sulfonic acid or a reactivethiolester such as e.g. an S-(2-benzothiazolyl)thioester.

For the acylation of an amine of formula IIIb with a residue of asulfonic acid a reactive derivative of the sulfonic acid, e.g. asulfonyl chloride, is reacted with an amine IIIb.

The acylation of the amine of the general formula IIIb is optionallyperformed in the presence of a base such as sodium bicarbonate,potassium carbonate, triethylamine, pyridine or N-methyl morpholine inan inert solvent such as methylene chloride, chloroform,tetrahydrofuran, dioxane, acetonitrile or N,N-dimethylformamide. Thereaction temperature can vary in a wide range between about -50° C. and+100° C., preferably between about -20° C. and +50° C.

In accordance with variant d) of the process in accordance with theinvention a cyclic amine of formula IV is reacted with an oxo compoundof the formula B═O so as to obtain end products of formula I whereR^(7b) is the group --N═B. Compounds of formula B═O are e.g.

aldehydes, e.g. compounds of the general formula Y--CHO;

ketones, e.g. compounds of the general formula Y2CO: or

N-substituted oreides, e.g. compounds of the general formula

Y--NR¹⁰ --CR¹⁰ O

where Y and R¹⁰ are as above.

The said variant d) can be carried out in a manner known per se e.g. byreacting an aldehyde or a ketone corresponding to formula B═O with theamine of formula IV in an inert aprotic solvent, such as methylenechloride or toluene, and in the presence of an acidic catalyst such asp-toluenesulfonic acid and a water-binding agent such as molecularsieves or magnesium sulfate.

This reaction is preferably carried out at a temperature in a range ofabout 0°-60° C.

When the compound of the formula B═O is the above mentionedN-substituted amide, the latter is reacted via the corresponding iminoylhalide, preferably the iminoyl chloride, e.g. by treatment of saidN-substituted amide with a chlorinating agent such as phosphoruspentachloride or phosphorus oxychloride and subsequently with the amineof formula IV. This reaction is carried out in an inert solvent such asmethylene chloride or tetrahydrofuran and in the presence of a base suchas triethylamine or pyridine. The reaction temperature preferably liesin the range of about -20° C. to +50° C.

In accordance with variant e) of the process in accordance with theinvention a carboxylic acid of the formula V is esterified or acarboxylic acid of the formula I is amidated with an agent yielding thecorresponding ester or amide moiety. For example, esterification may beaccomplished by treatment of the carboxylic acid of formula V or areactive derivative thereof, such as the corresponding acid chloride,acid anhydride, N-hydroxy-succinimide ester or methyl ester

--with an alcohol of the general formula Y--OH; whereas amidation can beeffected by treatment of a carboxylic acid of formula V

--with an amine of the general formula NH₂ R¹⁰,NH(OH)R¹⁰ or YNHR¹⁰,where Y and R¹⁰ are as above.

If the carboxylic acid of the formula V is reacted directly, i.e.without previous transformation into a reactive derivative, with analcohol Y--OH (carboxylic acid V only) or with an amine NH₂R¹⁰,NH(OH)R²⁰ or Y--NHR¹⁰, a coupling agent such as a carbodiimide, e.g.clicyclohexylcarbodiimide, or a 1-lower alkyl-2-halopyridinium salt,e.g. 1-methyl-2-chloropyridinium iodide, should be used.

These esterification and amidation reactions are preferably carried outin an inert solvent such as methylene chloride, tetrahydrefuran oracetonitrile and at a temperature in the range of about -20° C. to +80°C.

According to variant f) of the process in accordance with the inventiona starting compound of formula VI which is protected at any of amino,hydroxy and/or carboxy groups present) is deprotected to yield acompound of formula I with free amino, hydroxy and/or carboxy groups.

Possible amino-protecting groups are those employed in peptidechemistry, such as an alkoxycarbonyl group, e.g., t-butoxycarbonyl,etc., a substituted alkoxycarbonyl group, e.g., trichloroethoxycarbonyl,etc., a substituted aralkyloxycarbonyl group, e.g.,p-nitrobenzyloxycarbonyl, an aralkyl group such as trityl or benzhydrylor a halogen-alkanoyl group such as chloroacetyl, bromoacetyl ortrifiuoroacetyl.

Preferred amino-protecting groups are t-butoxycarbonyl, trityl and2,2,2-trichloroethoxycarbonyl.

The amino protecting groups may be cleaved off by acid hydrolysis (e.g.the t-butoxycarbonyl or trityl group) or by basic hydrolysis (e.g. thetrifiuoroacetyl group). The chloroacetyl, bromoacetyl and iodoacetylgroups are cleaved off by treatment with thiourea. The2,2,2-trichloroethoxycarbonyl group is cleaved off by reduction withzinc and an acid.

Amino-protecting groups which are cleavable by acid hydrolysis arepreferably removed with the aid of a lower alkanecarboxylic add whichmay be halogenated. In particular, formic acid or trifiuoroacetic add isused. The acid hydrolysis is generally carried out at room temperature,although it can be carried out at a slightly higher or slightly lowertemperature (e.g. at a temperature in the range of about 0° C. to +40°C.). Protecting groups which are cleavable under basic conditions aregenerally hydrolyzed with dilute aqueous alkali at 0° C. to +30° C. Thechloroacetyl and bromoacetyl protecting groups can be cleaved off usingthiourea in acidic, neutral or alkaline medium at about 0°-30° C. The2,2,2-trichloroalkoxycarbonyl group is cleaved by treatment with zincand an add, preferably aqueous acetic acid.

Possible hydroxy-protecting groups are the easily cleavable groups R⁴defined above, e.g. lower alkanoyl and lower alkoxycarbonyl. They may becleaved by basic hydrolysis, e.g. treatment with an inoganic base suchas an alkali metal hydroxide or carbonate in a lower alkanol, e.g.methanol, or tetrahydrofuran and at a temperature in the range of about0° C. and room temperature. The aforesaid silyl groups can be cleaved bytreatment with fluoride ions, e.g. by using ammonium fluoride ortetrabutylammonium fluoride in a solvent such as methanol ortetrahydrofuran, preferably at about 0° to room temperature. Hydroxygroups may further be protected or masked as acetals or ketals, e.g. astetrahydropyranyl ethers or methoxymethyl ethers. From these protectiongroups the hydroxy function can be liberated by acidic hydrolysis. e.g.with aqueous hydrochloric acid.

As carboxy-protecting groups one may utilize an ester form which can beeasily converted into a free carboxyl group under mild conditions, thecarboxy-protecting group being exemplified by, for example, t-butyl,p-nitrobenzyl, benzhydryl, allyl, 2,2,2-trichloroethyl etc. For example,the following reagents and their corresponding compatible esters areutilized: p-nitrobenzyl can be removed by hydrolysis in the presence ofsodium sulfide at about or below 0° C. to room temperature in a solvent,such as, dimethylformamide (aqueous); t-butyl can be removed by reactionwith trifiuoroacetic acid, optionally in the presence of anisole atabout 0° C. to room temperature with or without a co-solvent, such asmethylene chloride: allyl can be removed by a palladium (O) catalyzedtransallylation reaction in the presence of sodium or potassium salt of2-ethyl hexanoic acid, see for example J. Org. Chem. 1982, 47,587;2,2,2-trichloroethyl can be removed by reaction with zinc in a solventsuch as aqueous acetic acid or a mixture of tetrahydrofuran and aqueoussodium dihydrogen phosphate.

The residues of in vivo easily cleavable esters may also be employed ascarboxy-protecting groups. Examples of such esters, which can be of theconventional type, are the lower alkanoyloxalkyl esters (e.g., theacetoxymethyl, pivaloyloxymethyl, 1-acetoxyethyl and 1-pivaloyloxyethylester), the lower alkoxycarbonyloxyalkyl esters (e.g., themethoxycarbonyloxymethyl, 1-ethoxycarbonyloxyethyl and1-isopropoxycarbonyloxyethyl ester). These easily cleavable estersgroups may be split off by treatment with an esterase such as pig liveresterase in aqueous solution in the presence of a co-solvent such astetrahydrofuran or dimethylsulfoxide and at a temperature in the rangeof about 30° C. and 40° C.

Also conventional lower alkyl groups, e.g. methyl and ethyl, are usefulas carboxy-protecting groups: they can be split off in the same manneras the lower alkanoyl and lower alkoxycarbonyl groups R⁴ describedabove. Thus, treatment with an inorganic base such as an alkali metalhydroxide or carbonate in a lower alkanol or tetrahydrofuran at about 0°C. to room temperature will remove these hydroxy and carboxy-protectinggroups.

In the process variants a) to e) aforesaid R⁵, R^(7b), R^(70b), R^(71b)and R⁸ preferably do not contain a thiocarbonyl group --CS-- and/or X²is preferably not a thiocarbonyl group --CS--, this due to the relativeinstability of such intermediates. For the manufacture of end productsof formula I containing such thiocarbonyl groups --CS-- one preferablyuses the corresponding carbonyl (--CO--) compound having any amino,hydroxy and/or carboxy group in the molecule protected as describedabove under f). The conversion --CO-- to --CS-- as a final step can thenbe accomplished by reaction with a thiation reagent such as phosphoruspentasulfide or, alternatively, with2,4-bis-(4-methoxyphenyl)-2,4-dithioxo-1,3,2,4-dithiadiphosphetane (seeTetrahedron 37, 3635 (1981), followed by the cleavage of any amino,hydroxy and/or carboxy protection groups as described above under f).

The oxidation of the starting compounds of formula I in which X¹ is--S-- according to embodiment g) yields the oxidized analogues offormula I wherein X¹ is --SO-- (sulfoxides). This oxidation is carriedout by using an organic or inorganic oxidizing agent. Various compoundswhich readily deliver oxygen can be used as the oxidizing agent; forexample, organic peroxides such as monosubstituted organic peroxides(e.g. C₁₋₄ alkyl- or alkanoylhydroperoxides such ast-butylhydroperoxide), performic acid and peracetic acid, as well asphenyl-substituted derivatives of these hydroperoxides such ascumenetiydroperoxide and perbenzoic acid. The phenyl substituent can, ifdesired, carry a further lower group (e.g. a lower alkyl or lower alkoxygroup), a halogen atom or a carboxy group (e.g. 4-methylperbenzoic acid,4-methoxy-perbenzoic acid, 3-chloroperbenzoic acid and mono-perphthalicacid). Various inorganic oxidizing agents can also be used as theoxidizing agent: for example, hydrogen peroxide, ozone, permanganatessuch as potassium or sodium permanganate, hypochlorites such as sodium,potassium or ammonium hypochlorite, peroxymonosulphuric andperoxydisulphuric acid. The use of 3-chloroperbenzoic acid is preferred.The oxidation is advantageously carried out in an inert solvent, forexample, in an aprotic inert solvent such as tetrahydrofuran, dioxane,methylene chloride, chloroform, ethyl acetate or acetone or in a proticsolvent such as water, a lower alkanol (e.g. methanol or ethanol) or alower alkanecarboxylic acid which may be halogenated (e.g. formic acid,acetic acid or trifiuoroacetic acid). The oxidation is generally carriedout at a temperature in the range of -20° C. to +50° C. In order toobtain the corresponding sulfoxide, i.e. a compound of formula I inwhich X¹ stands for --SO--, with substantial exclusion of thecorresponding sulfone (i.e. where X¹ is --SO₂ --) it is preferable touse the oxidizing agent in equimolar amounts or in slight excess inrelation to the starting material.

The manufacture of the pharmaceutically acceptable salts of thecompounds of formula I can be carried out in a manner known per se; forexample, by reacting a carboxylic acid of formula I with an equivalentamount of the desired base or, conversely, a free base of formula I withan organic or inorganic acid. The reaction is conveniently carded out ina solvent such as water or an organic solvent (e.g. ethanol, methanol,acetone and the like). The temperature at which the salt formation iscarded out is not critical. The salt formation is generally carded outat room temperature, but it can be carded out at a temperature slightlyabove or below room temperature, for example in the range of 0° C. to+50° C.

The acid addition salts can be converted into a free form by treatmentwith a base, such as a metal hydroxide, ammonia and the like, the basesalts are converted into the free form by treatment with an acid such ashydrochloric acid and the like.

The starting compounds of formulas II-VI can be prepared in accordancewith the following flow sheets 1 and 2:

Compounds IIa can be manufactured according to flow sheet 1 as follows:##STR15## Compounds IIb can be manufactured according to Flow Sheet 2 asfollows: ##STR16## In Flow Sheets 1 and 2 R¹, R⁴, R⁶, R^(7a) and R⁸ areas above and R²³, R³³, R⁵³ and R^(73b) are as R², R³, R⁵ and R^(7b),respectively, except that R²³, R³³ and R^(73b) can also be nitro andR²³, R³³, R⁵³ and R^(73b) can also contain a protected amino, hydroxyand/or carboxy group; Z¹ represents a carboxy protecting group, Z² ishydrogen or a hydroxy-protecting group and Z³ is hydrogen or an aminoprotecting group. X² is preferably --CO--; end products with X² ═--CS--are preferably obtained as a final step as described above under f).

Protecting groups Z¹, Z² and Z³ are such as are usually employed in theart for protecting carboxy, hydroxy or amino groups, respectively.Carboxy protecting groups Z¹ are e.g. allyl, p-nitrobenzyl or2,2,2-trichloroethyl. As protecting groups Z² and Z³ there come intoconsideration groups such as trityl, 2,2,2-trichloroethoxycarbonyl ort-butoxycarbonyl.

Besides the above mentioned easily cleavable hydroxy protecting groups,phenolic hydroxy groups of the intermediates VII and VIII can also beprotected as methyl ethers, i.e. the substituents R²³, R³³ and/or OR⁴can also represent a methoxy group. In a later phase of the synthesis,e.g. after formation of the ester VIII, these methoxy groups canoptionally be cleaved, e.g. using boron tribromide in methylene chlorideat a temperature between -80° C. and +20° C., and the free phenolicfunctions can be reprotected by protecting groups more suitable forcleavage in the final product (e.g. lower alkanoyl, loweralkoxycarbonyl, tri-lower alkylsilyl or di-phenyl(lower alkyl)silyl).

Reactions of Flow Sheet 1

The manufacture of the starting materials of formula lIa can proceedaccording to Flow Sheet I as follows:

A substituted o-toluic acid of formula VII is converted into a reactivederivative thereof, e.g. to the acid chloride by reaction with thionylchloride, oxalyl chloride or phosphoryl chloride, preferably in an inertsolvent such as methylene chloride, chloroform or toluene. The acidchloride so obtained is thereafter reacted with an alcohol of thegeneral formula Z¹ OH, in which Z¹ is as above, e.g. with2,2,2-trichloroethanol, preferably in an inert solvent such as methylenechloride or tetrahydrofuran in the presence of an organic base such astriethylamine or pyridine, to yield the desired ester of formula VIII.

The ester of formula VIII is converted into a substituted benzyl bromideof formula IX by bromination e.g. by reaction with N-bromosuccinimide inrefluxing carbontetrachloride in the presence of a radical starter suchas α,α'-azo-isobutyronitrile or under irradiation with light, oralternatively by treatment with bromine in an inert solvent such ascarbontetrachloride under irradiation with light.

The so obtained substituted benzyl bromide of formula IX can becondensed with a thiol of formula XII to yield a thioether of formulaXIV. The reaction is preferably effected in an inert organic solventsuch as methylene chloride, ethyl acetate, N,N-dimethylformamide,dimethylsulfoxide, acetonitrile or ethanol in the presence of a weaknon-nucleophilic organic base such as triethylamine, or in the presenceof an inorganic base such as sodium or potassium carbonate. The reactiontemperature preferably lies between -60° C. and +60° C., preferablybetween 0° C. and +30° C.

The thiol of formula XII can be obtained by amidating a carboxylic acidof formula XI with an aminothiol of formula X in a manner commonly knownin peptide chemistry. According to a particularly preferred methodcompounds X and XI are reacted with each other in the presence of acondensation agent such as N,N'-dicyclohexylcarbodiimide, preferably inan aprotic organic solvent, such as acetonitrile, dioxane or methylenechloride, and at a temperature between -20° C. and +20° C., preferablyat -10° C. to +10° C. The aminothiol of formula X can be utilized asbase or as salt with an inorganic or organic acid, e.g. ashydrochloride; in the latter case an organic base such asN-methylmorpholine need be added in the reaction, preferably inequimolar amount.

An alternative method for obtaining the thioether of formula XIVconsists in reacting the substituted benzyl bromide of formula IX withthe aminothiol of formula Xa (substantially as described for thecondensation of compounds IX and XII). The so obtained amine of formulaXIII is thereafter reacted with the carboxylic acid of formula XI(substantially as described above for the amidation reaction involvingcompounds X and XI).

The starting compound of formula IIa, where X¹ is --S--, is obtained bycleavage of the protecting groups Z¹ and Z² in the thioether of formulaXIV so obtained. This is carried out as already described above for thedeprotection of starting compounds V according to variant f).

By oxidizing a thioether of formula XIV obtained in the manner describedabove for process alternative g), e.g. by oxidizing with3-chloroperbenzoic acid in methylene chloride and subsequently splittingoff the protecting groups Z¹ and Z² the corresponding sulfoxide, i.e.starting compounds of formula II, wherein X¹ is --SO--, are obtained.

Reactions of Flow Sheet 2

The manufacture of the starting compounds IIb employed as startingmaterials can proceed according to flow sheet 2 as follows:

A substituted o-toluic acid of formula VII is converted to the compoundof formula XV in analogy to the above conversion of compounds VII toesters of formula VIII. In so doing, the intermediate carboxylic acidchloride is reacted with an alcohol of formula XIa.

The conversion of the compound of formula XV obtained into thesubstituted benzyl bromide of formula XVI is effected by bromination inanalogy to the conversion of the ester VIII into the substituted benzylbromide of formula IX.

The substituted benzyl bromide of formula XVI can be condensed with athiol of formula Xa to yield a thioether of formula XIX in accordancewith the method described above for the manufacture of compounds XIVfrom compounds of formula XII.

The thioether of formula XIX can alternatively be obtained byesterification or amidation of a carboxylic acid of formula XVIII withan alcohol of formula XIa suitably employing a condensation agent suchas N,N'-dicyclohexylcarbodiimide, and in an inert organic solvent suchas acetonitrile or dioxane.

The thioether of formula XVIII employed in the above reaction can beobtained by reacting a substituted benzyl bromide of formula IX with athiol of formula Xa (substantially in accordance with the abovedescribed manufacture of compounds XIV from compounds XII). Thethioether of formula XVII so obtained is subsequently submitted to estercleavage (the ester protection group Z¹ being split off). The estercleavage is carried out in a manner known per se; when Z¹ is2,2,2-trichloroethyl the cleavage can be carried out by treatment withzinc powder in aqueous acetic acid.

For the manufacture of starting compounds lib, where X¹ is --S--, theprotecting groups Z¹ and Z² in the intermediate of formula XIX obtainedare cleaved off in a manner known per se, e.g. as described above. Byoxidizing intermediates of formula XIX in a manner described accordingprocess alternative g) above and subsequently cleaving off theprotecting groups Z¹ and Z² starting compounds of formula IIb, whereinX¹ is --SO--, are obtained.

It has also been found that one derivative falling within the generalformula I, viz. the compound of formula ##STR17## i.e.N-[[(4R,7S)-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy7-[[(3R)-3-hydroxy-1-L-seryl-L-prolyl)]amino]-11-methyl-6,10-dioxo-9,2,5-benzooxathiaazacyclododecin-4-yl]carbonyl]-L-alanine,is obtainable by fementation, more particularly by cultivating asubculture of the microorganism Streptomyces sp. NR0484 FERM-BP-1982under submerged, aerobic conditions in an aqueous carbohydrate solutioncontaining nitrogenous nutrients and sodium chloride, thereafterisolating the desired compound from the fermentation broth and, ifdesired, converting the product obtained into a pharmaceuticallyacceptable salt thereof, e.g. the sodium salt.

The organism producing compound Ia is a new strain designatedStreptomyces sp. NR0484. The organism given the laboratory designationNR0484 was deposited on Jul. 27, 1988, at Fementation Research Institute(FRI), 1-3, Higashi I-chome, Yatabe-machi. Tsukuba-gun, ibaraki-ken 305,Japan, under the Budapest Treaty and added to its collection ofmicroorganisms as FERM-BP-1982. The culture has been identified as astrain of Streptomyces filipinensis.

Strain NR0484 was isolated from a sandy soil sample collected atOhsemachi Hamamatsu City, Shizuoka, Japan by directly spreading ontothan potato carrot agar medium (Higgins, M. L., M. P. Lechevalier and H.A. Lechevalier: Flagellated actinomycetes, J. Bacteriol. 93:1446-1451,1967) supplemented with 10% grated radish. Strain NR⁰⁴⁸⁴ was directlycompared with the type strain (IF012860)of Streptomyces filipinensis.

The taxonomic studies were done according to the procedure adopted bythe International Streptomyces Project (ISP) using the media recommendedby Shirling, E. B. and D. Gottlieb (Method for characterization ofStreptomyces species; Intern. J. Syst. Bact. 16:313-340, 1966) and byWaksman, S. A. (Classification, identification and description of generaand species; in The Actinomycetes Vol. II; The Williams and Wilkins Co.,Baltimore, 1961). All cultures were incubated at 27° C. for 15 days. Thecolour scheme in the Colour Harmony Manual (4th ed., 1958; ContainerCorporation of America, Chicago) was used to describe the pigmentationof the colonies. Sodium chloride tolerance of the strains and theirgrowth at various temperatures were tested on ISP medium No. 3 (Difco)containing 0.15% yeast extract; and streptomycin sensitivity of thestrains were tested on ISP medium No. Z (Difco).

Cells for chemotaxonomic analyses were obtained from cultures inyeast-glucose broth (containing 1% yeast extract and 1% glucose, pH7.2). The analysis of whole cell hydrolyzates was done according to thethin-layer chromatographic procedure of Staneck, J. I. and G. D. Roberts(simplified approach to identification of aerobic actinomycetes bythin-layer chromatography; Appl. Microbiol. 28:226-231, 1974). The cellwall preparation was obtained from washed mycelia by the method ofYamaguchi, T. (comparison of the cell wall composition ofmorphologically distinct actinomycetes, J. Bacteriol. 89:444-453, 1965).The procedure of Becker, B., M. P. Lechevalier and H. A. Lechevalier(Chemical composition of cell-wall preparations from strains of variousform-genera of aerobic actinomycetes; Appl. Microbiol. 13:236-243, 1965)was used to identify amino acids in the cell wall.

Description of Strain NR0484 Morphological and chemical characteristics

Strain NR0484 grew well on agar media of various compositions showingcharacteristic growth for Streptomycetes and formed aerial mycelia. Thespore chain morphology consisted of spirals which could be categorizedas spirales. More than 50 oval to cylindrical mature sporechains wereformed. The surface of the spores were spiny and their dimensions were0.6-0-0.9×0.9-1.2 μm.

Whole cell hydrolysates of the strain contained LL-diaminopimelic acid,but not meso-diaminopimelic acid, and glycine was detected in the cellwall. Thus, the cell wall type of this strain is chemotype I ofLechevalier, M. P. and H. A. Lechevalier (Chemical composition as acriterion in the classification of aerobic actinomycetes; Intern. J.Syst. Bact. 20:435-443, 1970).

Based on the morphology and chemical characteristics of strain NR0484,this organism was assigned to the genus Streptomyces.

Cultural Characteristics

The growth characteristics of strain NR0484, cultivated for 15 days at27° C., on various agar media are summarized in Table 1. Media ISP 2, 3,4, 5 and 7 supported the best growth and abundant sporulation. Thecolour of aerial mycelia matched that of the Gray colour series, whereasthe colour of diffusible pigment could not be distinguished.

                  TABLE 1                                                         ______________________________________                                        Cultural characteristics of strain NR0484                                     Medium         Cultural characteristics                                       ______________________________________                                        Sucrose nitrate agar                                                                         G: moderate, colourless                                        (Waksman medium No. 1)                                                                       AM: thin, beige (3ge)                                                         R: beige (3ge)                                                                DP: none                                                       Yeast ext.-malt ext. agar                                                                    G: good, colourless                                            (ISP medium No. 2)                                                                           AM: good, ashes (5fe)                                                         R: clove braun (3ni)                                                          DP: none                                                       Oatmeal agar   G: good, yellow maple (3ng)                                    (ISP medium No. 3)                                                                           AM: good, ashes (5fe)                                                         R: beaver (3li)                                                               DP: none                                                       Inorganic salts-starch agar                                                                  G: good, yellow maple (3ng)                                    (ISP medium No. 4)                                                                           AM: good, ashes (5fe)                                                         R: adobe brown (31g)                                                          DP: none                                                       Glycerol asparagine agar                                                                     G: good, beaver (3li)                                          (ISP medium No. 5)                                                                           AM: good, ashes (5fe)                                                         R: beaver (3li)                                                               DP: none                                                       Glucose asparagine agar                                                                      G: moderate, amber (31c)                                       (Waksman medium No. 2)                                                                       AM: thin, pussywillow gray (5dc)                                              R: amber (31c)                                                                DP: none                                                       Tyrosine agar  G: good, glove brown (3ni)                                     (ISP medium No. 7)                                                                           AM: good, ashes (5fe)                                                         R: clove brown (3ni)                                                          DP: none                                                       Nutrient agar  G: moderate, colourless                                        (Waksman medium No. 14)                                                                      AM: thin, alabaster tint (13ba)                                               R: clove brown (3ni)                                                          DP: none                                                       ______________________________________                                          G: Growth                                                                    AM: Aerial mycelium                                                           R: Reverse                                                                    DP: Diffusible pigment                                                   

Physiological Characteristics

The carbon utilization and other physiological characteristics of strainNR0484 as well as Streptomyces filipinensis IFO 12860 are shown inTables 2 and 3. Both strains utilized carbohydrates similarly and hadsimilar physiological properties.

Species Determination

When strain NR0484 was compared with Streptomyces species described inthe literature, S. filipinensis (Atomann, A., D. Gottlieb, T. D. Brock,H. E. Carter and G. B. Whitfield: Filipin, an antibiotic effectiveagainst fungi; Phytopathology, 45:559-563, 1955) most closely resembledstrain NR0484. Therefore, the microbiological characteristics of strainNR0484 were directly compared with those of S. filipinensis IFO 12860,and it was found that strain IFO 12860 grown in the same productionmedium differed from strain NR0484 in that it could not produceDNA-gyrase inhibitor compound Ia. In spite of this minor difference,strain NR0484 and strain IFO12860 showed numerous similarities in otherproperties including morphology and cultural characteristics. Thus, itwas concluded that strain NR0484 could be assigned to the speciesStreptomyces filipinensis. For the purpose of the present inventionStreptomyces sp. NR0484 includes all strains of Streptomyces which formcompound Ia and which cannot be definitely differentiated from theculture numer NR0484 and its subcultures, including mutants andvariants. Compound Ia is identified herein and after this identificationis known, it is easy to differentiate the strains producing compound Iafrom others.

                  TABLE 2                                                         ______________________________________                                        Carbohydrate utilization by strain NR0484 and Streptomyces                    filipinensis IFO12860                                                                  strain NR0484                                                                           S. filipinensis IFO12860                                   ______________________________________                                        L-Arabinose                                                                              +           +                                                      D-Xylose   +           +                                                      G-Glucose  +           +                                                      D-Fructose +           +                                                      Sucrose    ±        +                                                      Inositol   ±        +                                                      L-Rhamnose ±        ±                                                   Raffinose  ±        +                                                      D-Mannitol +           +                                                      ______________________________________                                         +: Utilization                                                                ±: Probable utilization                                               

                  TABLE 3                                                         ______________________________________                                        Physiological characteristics of strain NR0484 and Streptomyces               filipinensis IFO12860                                                                                   S. filipinensis                                                    strain NR0484                                                                            IFO12860                                            ______________________________________                                        Gelatin liquefaction                                                                           -            -                                               (Waksman medium No. 19)                                                       Starch hydrolysis                                                                              +            +                                               (ISP medium No. 4)                                                            Milk coagulation -            -                                               (Difco 10% skimmed milk)                                                      Milk peptonization                                                                             +            +                                               (Difco 10% skimmed milk)                                                      Nitrate reduction                                                                              -            -                                               (ISP medium No. 8)                                                            Melanoid pigment formation                                                    ISP medium No. 1 +            +                                               ISP medium No. 6 +            +                                               ISP medium No. 7 -            -                                               NaCl tolerance   >4%,         >4%,                                                             but <10%     but <10%                                        Streptomycin sensitivity                                                                       6.3          6.3                                             (M.I.C., mg/ml)                                                               Temperature range for growth                                                                   20-45° C.                                                                           20-45° C.                                Optimum temperature for                                                                        30-37° C.                                                                           30-37° C.                                growth                                                                        Production of DNA-gyrase                                                                       +            -                                               inhibitor in A24 medium                                                       ______________________________________                                         +: Positive                                                                   -: Negative                                                              

Effect of Carbon and Nitrogen Sources

Five carbon sources and 21 nitrogen sources were tested for theproduction of compound Ia. Glucose was the best carbon source and yeastextract supplemented with-soybean meal (Toast soya, Nishin Seiyu) wasthe best nitrogen source.

Effect of Metal Ions

Following the studies on carbon and nitrogen sources, the influence ofCaCO₃ and NaCl was investigated. NaCl was necessary for the productionof compound Ia, whereas CaCO₃ caused less coproduct(s) to be made.

Based on the data described above, a fermentation medium, named A24medium consisting of 2% glucose. 2% Toast soya, 0.5% yeast extract,0.25% NaCl, 0.005% ZnSO₄ ·7H₂ O, 0.0005% CuSO₄ ·5H₂ O and 0.0005% MnCl₂·4H₂ O was selected as preferred fermentation medium.

Streptomyces sp. NR0484, when grown under suitable conditions, producesa compound of the formula Ia. A fermentation broth containingStreptomyces sp. NR0484 is prepared by inoculating spores or mycelia ofthe organism producing compound Ia into a suitable medium and thencultivating under aerobic conditions. For the production of compound Iacultivation on a solid medium is possible, but for production in largequantities cultivation in a liquid medium is preferable. The temperatureof the cultivation may be varied over a wide range, 20°-35° C., withinwhich the organism may grow, but a temperature of 26°-30° C. and asubstantially neutral pH are preferred. In the submerged aerobicfermentation of the organism for the production of compound Ia themedium may contain as the source for carbon a commercially availableglyceride oil or a carbohydrate such as glycerol, glucose, maltose,lactose, dextrin, starch etc. in pure or crude states, and as the sourceof nitrogen an organic material such as soybean meal, distillers'solubles, peanut meal, cotton seed meal, meat extract, peptone, fishmeal, yeast extract, corn steep liquor etc. and when desired inorganicsources of nitrogen such as nitrates and ammonium salts. Mineral saltpresent is sodium chloride, optionally also e.g. ammonium sulfate,magnesnium sulfate, potassium chloride, potassium phosphate, calciumcarbonate and trace mounts of heavy metal salts. The medium may alsocontain buffering agent such as sodium citrate or phosphates. In aeratedsubmerged culturing procedures, an anti-foam agent, such as liquidparaffin, fatty oils or silicone compounds, is preferably used. Morethan one kind of carbon source, nitrogen .source or anti-foam source maybe used for production of compound Ia.

After termination of fermentation compound Ia or its salts can berecovered from the fermentation medium. This can be accomplished by acombination of a variety of methods, for example, 1) adsorption on aadsorbent such as activated charcol and Diaion HP-21 (MitsubishiChemical Ind., Tokyo), 2) column chromatography on ion exchange resinsuch as Amberlite CG-50, IRC-50, DEAE-Toyopearl, QAE-Toyopeafi (TOSOH,Tokyo), DEAE-Sephadex and QAE-Sephadex, 3) reverse phase columnchromatography, 4) gel filtration using e.g. Toyopeafi HW-40, SephadexLH-20, Sephadex G-10, Sephadex G-15 and Sephadex G-25.

The compounds of formula I as well as their correspondingpharmaceutically acceptable salts inhibit DNA gyrase activity inbacteria and possess antibiotic, especially bactericidal activityagainst microorganisms.

A. Inhibition of DNA Gyrase Activity

The inhibition of DNA gyrase activity was measured using a DNA gyrasesupercoiling assay according to R. Otter & N. Cozzarelli:Methods inEnzymology, Vol. 100, pp 171-180 (1983). DNA gyrase was isolated from E.coli H560, and relaxed pUC18 plasmid was used as substrate. Theactivities as regards inhibition of DNA gyrase activity, expressed asmaximum non-effective concentration of the test compound (MNC in μg/ml)are compiled in the following Table 4:

                  TABLE 4                                                         ______________________________________                                        End product from                                                              Example No.     MNC (μg/ml)                                                ______________________________________                                         2              0.1                                                            3              5                                                              4              1                                                              5              10                                                             6              2                                                              9              1                                                             10              10                                                            12              5                                                             13              0.05                                                          41              0.1                                                           69              0.05                                                          84              0.1                                                           101             0.1                                                           102             0.25                                                          137             0.2                                                           ______________________________________                                    

B. Antibacterial Activity in Vitro

In the following table there are compiled the minimum inhibitoryconcentrations (MIC; μg/ml) of some representative compounds of formulaI against a series of pathogenic microorganisms.

    ______________________________________                                                   MIC (μg/ml)                                                                Compound of Example                                                Organism     41     69     84    101   102  137                               ______________________________________                                        E. coli DC2  64     16     >128  64    64   64                                E. coli B    128    32     >128  64    64   64                                N. meningitidis 69480                                                                      8      2      4     8     2    8                                 B. catarrhalis RA 117                                                                      4      4      4     4     2    4                                 S. aureus 887                                                                              32     32     16    16    8    32                                S. aureus 25923                                                                            32     128    32    16    16   128                               S. pyogenes 15                                                                             16     4      16    16    8    8                                 S. faecalis 6                                                                              32     16     32    32    16   64                                ______________________________________                                    

Agar dilution (BB2+1% Isovitalex+7.5% sheep blood+metadione), Inoculum:10⁴ CFU/spot.

The products in accordance with the invention can be used asmedicaments, e.g. in the form of pharmaceutical preparations for enteralor parenteral application. They can be administered, for example,perorally, e.g. in the form of tablets, coated tablets, dragees, hardand soft gelatine capsules, solutions, emulsions or suspensions,rectally, e.g. in the form of suppositories, or parenterally e.g. in theform of injection solutions.

The manufacture of the pharmaceutical preparations can be effected in amanner which is familiar to any person skilled in the art by bringingthe substances in accordance with the invention, optionally incombination with other therapeutically valuable substances, into agalenical administration form together with suitable, non-toxic, inert,therapeutically compatible solid or liquid carrier materials and, ifdesired, the usual pharmaceutical adjuvants.

As such carrier materials not only inorganic carrier materials aresuitable, but also organic carrier materials. Thus, there can be used ascarrier materials for tablets, coated tablets, dragees and hard gelatinecapsules, for example, lactose, maize starch or derivatives thereof,talc, stearic acid or its salts. Suitable carriers for soft gelatinecapsules are, for example, vegetable oils, waxes, fats and semi-solidand liquid polyols (depending on the nature of the active substance nocarriers are, however, required in the case of soft gelatine capsules).Suitable carder materials for the manufacture of solutions and syrupsare, for example, water, polyols, saccharose, invert sugar and glucose.Suitable carder materials for injection solutions are, for example,water, alcohols, polyols, glycerine and vegetable oils. Suitable cardermaterials for suppositories are, for example, natural or hardened oils,waxes, fats and semi-liquid or liquid polyols. The pharmaceuticalpreparations can also contain other therpeutically valuable substances.

As pharmaceutical adjuvants there come into consideration the usualpreservatives, solubilizers, stabilizers, wetting agents, emulsifiers,sweeteners, colorants, flavorants, salts for varying the osmoticpressure, buffers, coating agents and antioxidants.

The pharmaceutical preparations can contain the substances in accordancewith the invention in amounts of about 25-2000 mg, preferably 100-1000mg, per unit dosage form. For the prophylaxis and therapy of infectiousdiseases there comes into consideration for adults a daily dosage ofabout 0.05 g to about 4 g, especially about 0.1 g to about 2 g.

The following Examples are intended to illustrate the present inventionin more detail, but are not intended to limit its scope in any manner.

EXAMPLE 1

To 1.1 g of3,5-diacetoxy-6-[[(R)-2-((S)-2-(1-t-butoxyformamido)-3-hydroxypropionamido]-2-(methoxycarbonyl)ethyl)thio]methyl]-2-methylbenzoicacid in 19 ml of toluene were added at 0° C. 1.14 g2,2'-dithiobis(4-t-butyl-1-isopropylimidazole) and 0.74 g oftriphenylphosphine. After stirring for 40 minutes at 0° C., theheterogenous mixture was diluted with 80 ml of toluene. The wholemixture, while remaining cooled at 0° C. in a dropping funnel, was addedover a period of 2 hours to 40 ml of toluene which were kept at refluxtemperature. After complete addition, the reaction mixture was heated atreflux for another 3 hours. The mixture was cooled and the solventevaporated in vacuo. The residue was stirred in 30 ml of ethyl acetateat 0° C. for 1 hour and the crystalline4-t-butyl-N-isopropyl-2-mercaptoimidazole formed was removed byfiltration. The filtrate was evaporated in vacuo, and the residue waschromatographed using ethyl acetate/n-hexane (1:1, v/v) as eluent.Crystallization of the purified product from ethyl acetate/n-hexaneafforded t-butyl(4R,7S)-12,14-diacetoxy-1,3,4,5,6,7,8,10-octahydro-4-methoxycarbonyl-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamateas white crystals with mp 139° C. (decomposition).

¹ H-NMR (DMSO-d₆): δ1.41 (s,9H); 2.02 (s,3H); 2.31 (s,6H); 2.95 (dd,J=14Hz and 9 Hz,1H); 3.14 (dd,J=14 Hz and 4 Hz,1H); 3.43 (d,J=11 Hz,1H);3.63 (s,3H); 3.86 (d,J=11 Hz, 1H); 4.28 (dd,J=12 Hz and 4 Hz,1H);4.34-4.45 (m,1H); 4.51-4.14 (m,1H); 4.88-4.99 (m,1H); 7.12 (s,1H); 7.36(d,J=8 Hz,1H); 8.26 (d,J=8 Hz,1H) ppm

The starting material used above was prepared as follows:

(a) A solution of 10.75 g of 3,5-diimethoxy-2,6-dimethylbenzoic acid in10.7 ml of thionyl chloride was heated at reflux for 45 minutes, whengas evolution had ceased completely. The mixture was cooled to roomtemperature, whereupon a precipitate formed. Excess reagent wasevaporated in vacuo, 20 ml of toluene were added, and the mixture wasevaporated to dryness. The solid residue was dissolved in 50 ml ofmethylene chloride, and 9.2 g of 2,2,2-trichloroethanol were added tothe solution. The mixture was cooled to 0° C., and a solution of 6.2 gof triethylamine in 13 ml of methylene chloride was added within 10minutes. After stirring for ten minutes at 0° C. and for 3 hours at roomtemperature the reaction mixture was washed consecutively with 30 mlportions of 3N hydrochloric acid, water, saturated aqueous sodiumcarbonate solution and brine. The aqueous phases were backextracted with30 ml of methylene chloride. The organic layer was dried over sodiumsulphate and concentrated completely. The solid residue waschromatographed on 300 g of silica gel using acetone/n-hexane (1:6, v/v)for the elution. Crystallization of the purified product from n-hexaneafforded 2,2,2-trichloroethyl 3,5-dimethoxy-2,6-dimethylbenzoate aswhite crystals with mp 129° C.

(b) A solution of 2.73 g of 2,2,2-trichloroethyl3,5-dimethoxy-2,6-dimethylbenzoate in 8 ml of methylene chloride wascooled to -78° C., whereupon the dissolved material partly precipitated.To the stirred mixture a solution of 6.01 g of boron tribromide in 8 mlof methylene chloride was added dropwise within 20 minutes. During theaddition a clear solution formed, but later on a precipitate formedagain. After stirring for 10 minutes at -78° C. and for 2.5 hours at 0°C., the reaction mixture was poured on ice water with stirring, and theaqueous layer was extracted with methylene chloride. The organic layerwas washed with water, dried over sodium sulphate and evaporated todryness in vacuo. The residue was chromatographed on silica gel usingacetone/n-hexane (1:3, v/v) for the elution. Crystallization of thepurified product from ethyl acetate/n-hexane afforded2,2,2-trichloroethyl 3,5-dihydroxy-2,6-dimethylbenzoate as whitecrystals with mp 132°-133° C.

(c) A solution of 6.27 g of 2,2,2-trichloroethyl3,5-dihydroxy-2,6-dimethylbenzoate in 15 ml of acetic anhydride and 0.3ml of pyridine was heated under reflux for 3 hours. The solution wascooled and the solvent removed under reduced pressure. The residue wasdissolved in 20 ml of ethyl acetate and crystallized by the addition of120 ml of n-hexane to yield 2,2,2-trichloroethyl3,5-diacetoxy-2,6-dimethylbenzoate as white crystals with mp 127°-128°C.

(d) A mixture of 4.76 g 2,2,2-trichloroethyl3,5-diacetoxy-dimethylbenzoate and 2.24 g N-bromosuccinimide in 36 ml ofcarbontetrachloride was heated at reflux temperature and with lightirradiation for 30 minutes. The mixture was cooled in an ice bath andinsoluble material was removed by filtration. The filtrate was dilutedwith methylene chloride, washed with water, dried over sodium sulphateand evaporated in vacuo to provide crude 2,2,2-trichloroethyl3,5-diacetoxy-2-bromomethyl-6-methylbenzoate, which was used directlywithout further purification.

(e) A suspension of 17.2 g of L-cysteine methyl ester hydrochloride and20.5 g of N-(t-butoxycarbonyl)-L-serine in 300 ml of acetonitrile wastreated at 0° C. with 10.1 g of 4-methylmorpholine. To the stirredsolution was added dropwise at 10° C. within 30 minutes a solution of20.6 g N,N-dicyclohexylcarbodiimide in 300 ml of acetonitrile. Afterstirring the reaction mixture for 3 hours at 0° C. the precipitateformed was filtered off, and the filtrate was evaporated in vacuo. Theoily residue was dissolved in 200 ml of ethyl acetate and the solutionwashed consecutively with 0.5N hydrochloric acid, water, 5% aqueoussodium bicarbonate solution and brine. The organic layer was dried oversodium sulfate and evaporated in vacuo. The crude product waschromatographed on silica gel using ethyl acetate/n-hexane as eluent,and the so purified product was crystallized from diethyl ether/n-hexaneto give N-[N-(t-butoxycarbonyl)-L-seryl]-L-cysteine methyl ester aswhite crystals with mp 72°-74° C.

(f) To a solution of 3.8 g 2,2,2-trichloroethyl3,5-diacetoxy-2-bromomethyl-6-methylbenzoate and 2.9 g ofN-[N-(t-butoxycarbonyl)-L-seryl]-L-cysteine methyl ester in 30 ml ofmethylene chloride was added dropwise at 0° C. within 15 minutes asolution of 0.91 g of triethylamine in 5 ml of methylene chloride. Afterstirring for 1 hour at 0° C. and for 1 hour of 20° C., the reactionmixture was diluted with methylene chloride and washed with 1Nhydrochloric acid and with brine. The organic layer was dried oversodium sulfate and evaporated in vacuo. The remaining oil waschromatographed on silica gel using ethyl acetate/methylenechloride/n-hexane (1:1:1, v/v/v) as eluent. 2,2,2-Trichloroethyl3,5-diacetoxy-6-[[[CR)-2-[(S)-2-(1-t-butoxyformamido)-3-hydroxypropionamido]-2-(methoxycarbonyl)ethyl]thio]methyl]-2-methylbenzoatewas obtained as an oil.

¹ H-NMR (CDCl₃): δ1.46 (s,9H); 2.22 (s,3H); 2.33 (s,3H); 2.37 (s,3H);2.78 (dd,J=14 Hz and 6 Hz,1H); 2.87-2.95 (m,1H); 2.94 (dd,J=14 Hz and 4Hz,1H); 3.63-3.71 (m,1H); 3.73 (s,3H); 3.80 (s,2H); 4.05-4.17 (m,1H);4.19-4.27 (m,1H); 4.74-4.81 (m,1H); 4.97 (d,J=11 Hz,1H); 5.04 (d,J=11Hz,1H); 5.59 (d,J=7 Hz,1H); 7.01 (s,1H); 7.10 (d,J=7 Hz,1H) ppm.

(g) A mixture of 1.90 g of 2,2,2-trichloroethyl3,5-diacetoxy-6-[[((R)-2-[(S)-2-(1-t-butoxyformamido)-3-hydroxypropionamido]-2-(methoxycarbonyl)ethyl]thio]methyl]-2-methylbenzoate,50 ml of tetrahydrofuran, 12.5 ml of 1M phosphoric acid, 12.5 ml of 1Maqueous sodium dihydrogen phosphate solution and 3.2 g of zinc powderwas stirred at 20° C. for 1.2 hours. The mixture was filtered, and theunsoluble material was washed with water and ethyl acetate. The filtratewas partitioned between ethyl acetate and water. The organic layer wasdried over sodium sulfate and evaporated in vacuo to afford3,5-diacetoxy-6-[[[(R)-2-((S)-2-(1-t-butoxyformamido)-3-hydroxypropionamido]-2-(methoxycarbonyl)ethyl]thio]methyl]-2-methylbenzoicacid as a solidifying oil.

¹ H-NMR (DMSO-d₆): δ1.38 (s.9H); 2.05 (s,3H); 2.30 (s,3H); 2.31 (s,3H);2.74-2.96 (m,2H); 3.44-3.68 (m,2H); 3.62 (s,3H); 3.68 (s,2H); 3.98-4.12(m,1H); 4.54 (dd,J=14 Hz and 8 Hz,1H); 6.71 (d,J=8 Hz,1H); 7.06 (s,1H);8.27 (d,J=8 Hz,1H); 13.74 (broad s,1H) ppm

EXAMPLE 2

To a solution of 32 mg of t-butyl(4R,7S)-12,14-diacetoxy-1,3,4,5,6,7,8,10-octahydro-4-methoxycarbonyl-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamatein 3 ml of methanol were added 15 mg of potassium carbonate. Afterstirring for 30 minutes at room temperature the reaction mixture wasdiluted with ethyl acetate and washed consecutively with 0.1M pH 7aqueous sodium phosphate buffer and with brine. The organic layer wasdried over sodium sulfate and evaporated in vacuo. The residue waspurified by chromatography on silica gel using acetone/n-hexane 1:2(v/v) as eluent. The purified product was dissolved in methylenechloride and precipitated by slow addition of n-hexane to yield t-butyl(4R,7S)-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-4-methoxycarbonyl-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamate as a white, amorphousmaterial.

¹ H-NMR (CDCl₃): 1.50 (s,9H); 2.06 (s,3H); 2.93-3.25 (m,2H); 3.42(d,J=12 Hz.1H); 3.76 (s,3H); 3.96 (d,J=12 Hz,1H); 4.32-4.44 (m,1H);4.60-4.71 (m,1H); 4.84-4.95 (m1H); 5.17-5.30 (m,1H); 5.87 (d,J=8 Hz,1H);6.02 (broad s,1H); 6.43 (s,1H); 6.65 (broad s,1H); 7.37 (d,J=8 Hz,1H)ppm

EXAMPLE 3

To a solution of 284 mg of t-butyl(4R,7S)-12,14-diacetoxy-1,3,4,5,6,7,8,10-octahydro-4-methoxycarbonyl-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamate in 4.3 ml oftetrahydrofuran were added 1.4 ml of water. To the stirred mixture wereadded at room temperature within 1.5 hours 9 ml of 0.25N aqueous sodiumhydroxide solution in such a manner that the pH of the mixture was keptbetween 11.8 and 12.1. Stirring was continued for 2 hours at pH 11.8.The reaction mixture was extracted with ethyl ether and the organiclayer was back-extracted with 5% aqueous sodium bicarbonate solution.The combined aqueous solutions were acidified to pH 2 by the addition of3N aqueous hydrochloric add and extracted with ethyl acetate. The ethylacetate layer was washed with brine, dried over sodium sulfate andevaporated to dryness. The residue was crystallized from ethylacetate/n-hexane to yield(4R,7S)-7-(L-t-butoxyformamido)-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylic acid as a white powder,mp 176° C. (decomposition).

¹ H-NMR (DMSO-d₆): 1.41 (s,9H); 1.90 (s,3H); 2,90 (dd,J=15 Hz and 7.5Hz,1H); 3.04 (dd,J=15 Hz and 5 Hz,1H); 3.28 (d,J=10 Hz.1H); 3.91 d,J=10Hz,1H); 4.16 (dd.J=11 Hz and 3.5 Hz,1H); 4.24-4.37 (m,1H); 4.40-4.52m,1H); 4.92 (dd,J=11 Hz and 3.5 Hz); 6.44 s,1H); 7.39 (d,J=7HZ,1H); 7.90d,J=8 Hz,1H); 9.48 (s,1H); 9.50 (s,1H); 3.04 (broad s,1H) ppm

EXAMPLE 4

A solution of 117 mg of(4R,7S)-7-(1-t-butoxyformamido)-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylicacid in 2.5 ml methylene chloride/trifiuoroacetic acid (1:1, v/v) wasstirred at 0° C. for 1.5 hours. Ice was added and the pH of the mixturewas raised to 3 by the addition of 2N aqueous sodium hydroxide. Themixture was extracted with ethyl acetate and the organic layerback-extracted with water. The aqueous layer was concentrated in vacuoand the concentrated solution was chromatographed on MCI-Gel CHP20P(Mitsubishi Chemical Industries, Ltd.) using 1% aqueous acetic add aseluent. The product-containing fractions were combined and lyophilizedto give(4R,7S)-7-amino-1,3,4,5,6,7,8,10-octahydro-12,14-dihydro-11methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylic add as a white powder.

¹ H-NMR (DMSO-d₆): δ1.88 (s,3H); 2.91 (dd,J=14 Hz and 8 Hz,1H); 3.07(dd,J=14HZ and 6 Hz,1H); 3.26 (d,J=11 Hz,1H); 3.58-3.67 (m,1H); 3.87(d,J-11 Hz,1H); 4.06 (dd.J=9 Hz and 2 Hz,1H); 4.36-4.45 (m,1H); 5.12(dd,J=9 Hz and 1 Hz,1H); 6.43 (s,1H) ppm

EXAMPLE 5

A solution of 114 mg of t-butyl(4R,75)-12,14-diacetoxy-1,3,4,5,6,7,8,10-octahydro-4-methoxycarbonyl-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamate in 2.4 ml oftrifiuoroacetic acid was stirred at 0° C. for 30 minutes. The solutionwas poured onto a mixture of 20 ml of 0.5M aqueous sodiumdihydrogenphosphate solution, 4.7 ml of 28% aqueous sodium hydroxidesolution and crushed ice. The pH was adjusted to 9 by the addition ofsodium hydroxide and the mixture was then extracted with methylenechloride. The organic layer was washed with brine, dried over sodiumsulfate and evaporated in vacuo. The residue was dissolved in methylenechloride and crystallized by the addition of n-hexane to give methyl(4R,7S)-12,14-diacetoxy-7-amino-1,3,4,5,6,7,8,10-octahydro-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylateas a white powder.

¹ H-NMR (DMSO-d₆): δ2.00 (s,3H); 2.30 (s,3H); 2.31 (s,3H); 2.96 (dd,J=15Hz and 10 Hz,1H); 3.22 (dd,J=15 Hz and 5 Hz,1H); 3.39 (d,J=11 Hz,1H);3.62-3.76 (m, 1H): 3.66 (s,3H); 3.88 (d,J=11 Hz,1H): 4.22 (dd,J=11 Hzand 3 Hz,1H); 4.62-4.76 (m,1H); 5.16 (dd,J=1 Hz and 2 Hz,1H); 7.11(s,1H); 8.52 (d,J=8 Hz,1H) ppm

EXAMPLE 6

A solution of 14 mg of t-butyl(4R,7S)-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-4-methoxycarbonyl-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamatein 0.3 ml of trifiuoroacetic acid was stirred at 0° C. for 30 minutes.The solution was poured on ice, and the pH of the mixture was raised to3.5 by the addition of 1N aqueous sodium hydroxide solution. The clearsolution was chromatographed on MCI-Gel CHP20P using at first 0.1%aqueous acetic acid and then a mixture of 0.1% aqueous aceticacid/acetonitrile (9:1, v/v) as eluent. The fractions containing theproduct were lyophilized to give methyl(4R,7S)-7,amino-1,3,4,5,6,7,8,10-octahydro-12,14-dihydro-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylateas a white powder.

¹ H-NMR (DMSO-d₆): δ1.87 (s,3H) superposed by 1.90 (broad s,2H); 2.85(dd,J=15 Hz and 10 Hz,1H); 3.06 (dd,J=15 Hz and 4 Hz,1H); 3.39 (d,J=11Hz,1H); 3.66 (s,3H); 3.82 (d,J11 Hz,1H); 4.09 (dd,J=11 Hz and 3 Hz,1H);4.62 (m,1H); 5.11 (dd,J=11 Hz and 2 Hz,1H); 6.46 (s,1H); 8.45(d,J=8HZ,1H); 9.55 (broad s,2H) ppm

EXAMPLE 7

3,5-Diacetoxy-6-[[[2-[(5)-2-(1-t-butoxyformamido)-3-hydroxypropionamido]-2-ethyl]thio]methyl]-2-methylbenzoicacid was lactonized in analogous manner to the procedure described inExample 1 to afford, upon chromatographic purification andcrystallization from ethyl acetate/n-hexane, t-butyl(S)-12,14-diacetoxy-1,3,4,5,6,7,8,10-octahydro-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamate as a whitepowder with mp 233° C. (dec.).

¹ H-NMR (DMSO-d₆): δ1.39 (s,9H); 2.01 (s,3H); 2.30 (s,3H); 2.31 (s,3H):2.54-2.86 (m,2H); 3.02-3.16 (m,1H); 3.50-3.74 (m,3H); 4.30-4.49 (m,2H);4.564.68 (m,1H); 7.12 (s,1H); 7.17 (d,J=7 Hz,1H); 8.26-8.37 (m,1H) ppm

The starting material used above was prepared as follows:

(a) 2,2,2-Trichloroethyl 3,5-diacetoxy-2-bromomethyl-6-methylbenzoatewas reacted with t-butyl[(S)-2-hydroxy-1-[(2-mercaptoethyl)carbamoyl]ethyl]carbamate in a manneranalogous to the procedure given in Example 1(f), and the resulting2,2,2-trichloroethyl3,5-diacetoxy-6-[[((R)-2-[(S)-2-(t-butoxyformamido)-3-hydroxypropionamido]-2-ethyl]thio]methyl]-2-methylbenzoateproduct was subjected in analogous manner to the procedure described inExample 1(g) to give3,5-diacetoxy-6-[[[2-[(S)-2-(1-t-butoxyformamido)-3-hydroxypropionamido]-2-ethyl]thio]methyl]-2-methylbenzoicacid as an oil.

(b) t-Butyl [(S)-2-hydroxy-1-[(2-mercaptoethyl)carbamoyl]ethyl]carbamatewas obtained from cysteamine hydrochloride andN-(t-butoxycarbonyl)-L-serine in a manner analogous to the proceduredescribed in Example 1(e) as white crystals with mp 95°-96° C.

EXAMPLE 8

204 mg t-butyl(S)-12,14-diacetoxy-1,3,4,5,6,7,8,10-octahydro-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamatewas subjected to a procedure analogous to that described in Example 2.There was obtained, after crystallization from ethyl acetate/n-hexane,t-butyl (S)-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamate as a whitepowder with mp 215° C. (decomposition).

¹ H-NMR (DMSO-d₆): δ1.39 (s,9H); 1.89 (s,3H); 2.42-2.59 (m,1H);2.65-2.80 (m,1H); 2.98-3.15 (m,1H); 3.48-3.74 (m,3H); 4.26-4.55 (m,3H);6.44 (s,1H); 7.03 (d,J=6 Hz,1H); 8.20 (m,1H); 9.45 (s,1H); 9.49 (s,1H)ppm

EXAMPLE 9

64 mg t-butyl(S)-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamatewas subjected to a procedure analogous to that described in Example 6.There was obtained, after chromatographic purification andlyophilization, (S)-7-amino-4,5,7,8-tetrahydro-12,14-dihydroxy-11-methyl-9,2,5-benzoxathiaazacyclododecine-6,10(1H,3H)-dione as a white powder.

¹ H-NMR (DMSO-d₆): δ1.87 (s,3H) superimposed with 1.80-2.00 (broads,ca.4H); 2.42-2.80 (m,2H); 3.12-3.73 (overlapping m, ca. 8H); 4.09-4.26(m,1H); 4.46-4.76 (m,1H); 6.42 (s,1H); 8.08 (broad s,1H) ppm

EXAMPLE 10

To a suspension of 47 mg of methyl(4R,75)-12,14-diacetoxy-7-amino-1,3,4,5,6,7,8,10-octahydro-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacydododecine-4-carboxylatein 1 ml of acetonitrile were added 39 mg ofS-(2-benzothiiazolyl)-2-amino-4-thiazoleglyoxylate (Z)-O-methyloxime,and the mixture was stirred at 20° C. for 4 hours. The solvent wasevaporated in vacuo, and the residue was chromatographed on silica gelusing ethyl acetate/n-hexane (1:1, v/v) and ethyl acetate as eluent. Thefractions containing the product were evaporated and the residuecrystallized from methylene chloride/n-hexane,to give methyl(4R,7S)-12,14-diacetoxy-7-[(Z)-2-(2-amino-4-thiazolyl)-2-(methoxyimino)acetamido]-1,3,4,5,6,7,8,10-octahydro-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4carboxylate as a white powder.

¹ H-NMR (DMSO-d₆): δ2.02 (s,3H); 2.31 (s,6H); 2.90 (dd,J=14 Hz and 9Hz.1H); 3.24 (dd,J=14 Hz and 5 Hz,1H); 3.46 (d,J=12 Hz,1H); 3.65 (s,3H);3.83 (d,J=12 Hz,1H); 3.84 (s,3H); 4.28 (dd,J=11 Hz and 4 Hz,1H);4.52-4.61 (m,1H); 4.78-4.87 (m,1H); 5.12 (dd,J=11 Hz and 3 Hz,1H); 6.85(s,1H); 7.13 (s,1H); 7.20 (broad s,2H); 8.10 (d,J=8 Hz,1H); 9.44 (d,J=8Hz,1H) ppm

EXAMPLE 11

Methyl(4R,7S)-12,14-diacetoxy-7-[(Z)-2-(2-amino-4-thiazolyl)-2-(methoxyimino)acetamido]-1,3,4,5,6,7,8,10-octahydro-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylate, was subjected to a procedure analogous to that describedin Example 2. There was obtained, after crystallization from ethylacetate/n-hexane, methyl(4R,7S)-[(Z)-2-(2-amino-4-thiazolyl)-2-(methoxyimino)acetamido]-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylateas a white powder.

¹ H-NMR (DMSO-d₆): δ1.89 (s,3H); 2.78 (dd,J=14 Hz and 9 Hz,1H); 3.10(dd,J=14 Hz and 4.Hz,1H); 3.92 (d,J=11 Hz,1H); 3.64 (s,3H); 3.82 (d,J=11Hz,1H); 3.85 (s,3H); 4.15 (dd,J=11 Hz and 4 Hz,1H); 4.50-4.62 (m,1H);4.68-4.76 (m,1H); 5.60 (dd,J=11 Hz and 3 Hz,1H); 6.44 (s,1H); 6.83(s,1H); 7.16 (broad s,2H); 7.96 (d,J=8 Hz,1H); 9.38 (d,J=7 Hz,1H); 9.50(s,1H); 9.51 (s,1H) ppm

EXAMPLE 12 Tank Fermentation

The culture producing compound Ia was grown and maintained on an agarslant having the following composition (grams/liter distilled water):

    ______________________________________                                               Yeast extract                                                                          4                                                                    Malt extract                                                                           10                                                                   Glucose  4                                                                    Agar     20.0                                                                 (pH 7.3)                                                               ______________________________________                                    

The slant was inoculated with the culture and incubated at 27° C. for 14days. A chunk of the agar containing spores and mycelia from thesporulated culture slant was then used to inoculate a 500 ml Erlenmeyerflask containing 100 ml of sterilized inoculum medium (A24 medium)having the following composition (grams/liter distilled water):

    ______________________________________                                               Glucose  20.0                                                                 Toast soya                                                                             20.0                                                                 Yeast extract                                                                          5.0                                                                  NaCl     2.5                                                                  ZnSO.sub.4.7H.sub.2 O                                                                  0.05                                                                 CuSO.sub.4.5H.sub.2 O                                                                  0.005                                                                MnCl.sub.2.4H.sub.2 O                                                                  0.005                                                                (pH 7.0)                                                               ______________________________________                                    

The inoculated inoculum medium was incubated at 27° C. for 3 days on arotary shaker. A new 500 ml Eriemeyer flask containing 100 ml of thesame medium was inoculated with 2% of the first-stage inoculum andincubated on a rotary shaker for 3 days at 27° C.

The production-stage was done in a 200 1-fermentor coning 140 liters ofthe same medium which was inoculated with 2% of the second-stageinoculum, and operated at 27° C., agitated at 250 rpm, and aerated at140 liters/min with an inner pressure of 0.5 kg/cm². An antifoamingagent (Nissan disfoam) was added when there was a need. The broth washarvested afar 2 days.

Isolation

In the following isolation procedure, each fraction was monitored by DNAgyrase inhibition assay.

Harvested broth (140 liters) was separated to roycelia and filtrate.

Activated charcoal (1,100 g) was added to 110 liters of the filtrate.The mixture was stirred at room temperature for 30 minutes and thenfiltered. The carbon cake was then washed with water an suspended in 100liters (50 liters×2) of 50% aqueous acetone. After stirring at 60° C.for 30 min, the mixture was filtered, and the filtrate was concentratedunder reduced pressure.

The resulting brown syrup was dissolved in 500 ml of water and put on acolumn (10 liters)of Diaion HP-21 (Mitsubshi Chemical Industries, Tokyo)which was then eluted successively with water (20 liters) and 10%aqueous ethanol (50 liters). The active fractions eluted with 10%aqueous ethanol were pooled, the pooled sample was concentrated underreduced pressure and lyophilized to give 78,6 g of the residue as ayellow powder.

This lyophilized powder was then dissolved in 180 ml of water and put onan Amberlite CG-SO column (3.6 liters of a mixed bed consisting of onepart of ammonium form and five parts of H⁺ form, type 1).

The active fractions (1.8 liters) eluted with water were combined andput on a column of 2.5 liters of DEAE-Toyopearl, coarse type (Toyosoda),which was first eluted with water (12 liters) and then with 0.02M NaCl(80 liters). The fractions eluted with 0.02M NaCl were monitored by HighPressure Liquid Chromatography. The active fractions were combined (30liters) and put on a column of 2.5 liters of Diaion HP-21, and theneluted with water (5 liters) and after that with 50% aqueous acetone (15liters). The active fractions eluted with 50% aqueous acetone werecombined (10 liters) and concentrated to 100 ml under reduced pressureand partitioned between water and n-butyl alcohol. The aqueous layer wasconcentrated under reduced pressure and lyophilized to give crudecompound Ia as a pale yellow powder.

This material (303 mg) was dissolved in water (3 ml) and applied onto acolumn of 1.5 liters of Toyopearl HW-40, super fine type (Toyosoda), andeluted with water. Lyophilisation of the active fractions gave compoundIa as a pale yellow amorphous powder, i.e. N-[[(4R,7S)-1,3,4,5,6,78,10-octahydro-12,14-dihydroxy-7-[[(3R)-3-hydroxy-1-L-seryl-L-prolyl]amino]-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecin-4-yl]carbonyl]-L-alanine,probably in the form of its sodium salt.

The above is the correct chemical name.

The same compound was originally given the name of N-[[4R,7S-1,3,4,5,6,78,10-octahydro-12,14-dihydroxy-7-[(cis-3-hydroxy-1-S-seryl-prolyl)amino]-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecin-4-yl]carbonyl]-S-alanine.

Physicochemical Properties of Compound Ia

Compound Ia is an amphoteric, water soluble compound. Thephysicochemical properties of compound Ia are listed in Table 5.

The molecular formula of compound Ia was determined to be C₂₆ H₃₅ N₅ O₁₂S. Mass spectral data showed a protonated molecular ion (MH⁺) m/z642.2177 (calcd 642.2081); ¹ H and ¹³ C-NMR spectral data gave furtherevidence for this formula (Table 6).

                  TABLE 5                                                         ______________________________________                                        Physicochemical properties of compound Ia                                     Appearance   pale yellow amorphous powder                                     ______________________________________                                        [α].sub.D .sup.20                                                                    -13.2 (c 1.0, H.sub.2 O)                                         UV(H.sub.2 O)                                                                              293 (3.20)                                                       λmax (log ε).sub.nm                                            UV(H.sub.2 O+NaOH)                                                                         309 (3.30)                                                       λmax (log ε).sub.nm                                            IR (KBr)     3350 (NH, OH), 1720 (ester C═O),                             νmax cm.sup.-1                                                                          1640 (amide C═O)                                             HR-FAB-MS    Calcd: C.sub.26 H.sub.36 N.sub.5 O.sub.12 S (MH+) 642.2081                    Found: 642.2177                                                  Rf value     0.17 on TLC (silica gel)                                                      Solvent system:                                                               n-BuOH-AcOH-H.sub.2 O (4:1:2)                                    Colour reaction                                                                            ninhydrin (+), FeCl.sub.3 (+)                                    ______________________________________                                    

                  TABLE 6                                                         ______________________________________                                        .sup.1 H and .sup.13 C NMR chemical shifts and coupling constants for         compound Ia                                                                   in D.sub.2 O                                                                          Position                                                                      No.    δC                                                                              δH   J(H,H)                                      ______________________________________                                        Alanine   2        54.1    4.10(1H, q)                                                                            7.3 Hz                                    moiety    2-CH.sub.3                                                                             20.2    1.35(3H, d)                                                                            7.3 Hz                                                       182.5   --                                                 Cysteine  3        35.3    3.32(1H, d.d.)                                                                         15, 4.6 Hz                                moiety                     2.66(1H, d.d.)                                                                         15, 11.5 Hz                                         2        55.5    4.72(1H, m)                                                  1        173.4.sup.a)                                                                          --                                                 Serine(I) 3        65.8    3.70(1H, d.d.)                                                                         11.5, 5.5 Hz                              moiety                     3.58(1H, d.d.)                                                                         11.5, 6.5 Hz                                        2        56.3    3.88(1H, d.d.)                                                                         6.5, 5.5 Hz                                         1        173.4.sup.a)                                                                          --                                                 Serine(II)                                                                              3        66.6    5.66(1H, d.d.)                                                                         12, 2.4 Hz                                moiety                     4.36(1H, d.d.)                                                                         12, 2.2 Hz                                          2        58.0    4.72(1H, m)                                                  1        174.1a) --                                                 cis-3-Hydroxy-                                                                          5        48.2    3.80(2H, m)                                        proline   4        35.3    2.22(1H, m)                                        moiety                     2.05(1H, m)                                                  3        73.4    4.72(1H, m)                                                  2        66.6    4.72(1H, m)                                                  1        176.1                                                      Chromophore 1                                                                           137.5    --                                                         moiety.sup.b)                                                                           2        117.0   --                                                           3        159.2   --                                                           4        108.8   6.43(1H, s)                                                  5        157.6   --                                                           6        113.0   --                                                           2-CH.sub.3                                                                             14.7    1.98(3H, s)                                                  6-CH.sub.2                                                                             30.4    3.86(3H, s)                                                                            10.7 Hz                                                              3.46(1H, d)                                                                            10.7 Hz                                             1-CO     173.1   --                                                 ______________________________________                                         .sup.a) interchangeable                                                       .sup.b) Numbering of the chromophore:                                         ##STR18##                                                                

EXAMPLE 13

3,5-Bis(tert-butyldimethylsilyloxy)-6-[[[(R)-2-[(S)-1-tertbutoxyformamido)-3-hydroxypropionylamino]-2-methoxycarbonylethyl]thio]methyl]-2-methylbenzoicacid was lactonized in analogous manner to the procedure described inExample 1 to afford, upon chromatographic purification on silica gelusing ethyl acetate/hexane (1: 3, v/v) as eluent, tert-butyl (4R,7S)-12,14-bis(tert-butyldimethylsilyloxy)-1,3,4,5,6,7,8,10-octahydro-4-methoxycarbonyl-11-methyl-6,10-dioxo-9, 2,5-benzooxathiaazacyclododecine-7-carbamate as a paleyellow foam. To a solution of 21.4 g of this material in 300 ml ofmethanol were added 3.3 g of ammonium fluoride, and the mixture wasstirred for 30 minutes at room temperature. The solvent was evaporatedin vacuo. The residue chromatographed on silica gel using ethylacetate/hexane (1:1, v/v) as eluent, and the purified product wascrystallized from ethyl acetate/hexane to afford 11.0 g of tert-butyl(4R,7S)-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-4-methoxycarbonyl-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamate.

The starting material used above was prepared as follows:

(a) To a stirred mixture of 110 g of 37% aqueous formaldehyde, 250 ml ofethanol, and 250 ml of glacial acetic acid were added dropwise withcooling 126 g of 40% aqueous dimethylamine, keeping the temperature at-25©C. Stirring was continued for 30 minutes, whereupon the mixture wascooled to 10° C., and 200 g of 3,5-dihydroxy-2-methylbenzoic acid wereadded. The cooling bath was removed, and stirring was continued for 14hours. The white precipitate formed was isolated by filtration, washedwith water and dried to yield 150 g ofα-(dimethylamino)-3,5-dihydroxy-2,6-dimethylbenzoic acid acetate as awhite solid.

(b) A suspension of 150 g ofα-(dimethylamino)-3,5-dihydroxy-2,6-dimethylbenzoic acid acetate in 1.5l of methanol was treated with a suspension of 9 g of 5% palladium oncharcoal in 220 ml of 3N aqueous sodium hydroxide, and the mixture washydrogenated for 14 hours at a hydrogen pressure of 0.1 bar. The pH ofthe mixture was set to 1 by the addition of 37% hydrochloric acid, thecatalyst was filtered off, and the filtrate was concentrated in vacuo.The residue was diluted with 0.5 1 of water and then extracted with 1.21 of ethyl acetate. The organic layer was washed successively with 2Nhydrochloric acid and 15% sodium chloride solution, dried over sodiumsulfate and evaporated in vacuo to give, after crystallization fromdioxane, 87 g of 3, 5-dihydroxy-2,6-dimethylbenzoic acid as whitecrystals of m.p. 178°-179° C.

(c) To a solution of 91.1 g of 3,5-dihydroxy-2,6 -dimethylbenzoic acidand 108.0 g of p-nitrobenzyl bromide were added 165.9 g of potassiumcarbonate. The mixture was heated at reflux under stirring for 4 hours.After cooling to room temperature, the mixture was filtered, and theunsoluble material was washed with 0.5 l of ethyl acetate. The filtratewas diluted with 0.5 l of ethyl acetate and washed with ice-cold 3Nhydrochloric acid and with brine. The organic layer was dried oversodium sulfate and evaporated in vacuo. To the remaining, crystallizingoil were added 0.6 1 of tert-butylmethylether, and the suspension wasstirred for 1 hour. Unsoluble material was filtered off and the filtratewas evaporated in vacuo. The remaining oil was dissolved in 300 ml ofmethylene chloride. Upon crystallization, 200 ml of hexane were addedand the crystals isolated by filtration. This material was suspendedonce more in 400 ml of tert-butylmethylether and the unsoluble materialwas removed by filtration. Evaporation of the filtrate in vacuo andcrystallization of the residue from methylene chloride/hexane afforded98.8 g of p-nitrobenzyl 3,5-dihydroxy-2,6-dimethylbenzoate as yellowcrystals of m.p. 164°-166° C.

(d) To a mixture of 95.1 g of p-nitrobenzyl3,5-dihydroxy-2,6-dimethylbenzoate and 99.6 g oftert-butyldimethylchlorosilane in 240 ml of dimethylformamide, cooled to0° C., were added within 2 minutes 72.0 g of triethylamine, aprecipitate resulting immediately. The mixture was stirred at 0° C. for2 hours, then at room temperature for another 2 hours, and then dilutedwith 800 ml of ethyl acetate and washed 5 times with 400 ml of 15%sodium chloride solution. The aqueous phases were back-extracted with800 ml of ethyl acetate. The organic phases were dried over sodiumsulfate and evaporated in vacuo. Crystallization of the residualmaterial from 1.2 1 of hexane provided 141.8 g of p-nitrobenzyl3,5-bis(tert-butyldimethylsilyloxy)-2,6-dimethylbenzoate as colourlesscrystals of m.p. 121°-122° C.

(e) p-Nitrobenzyl3,5-bis(tert-butyldimethylsilyloxy)-2,6-dimethylbenzoate was subjectedin an analogous manner to the procedures described in Example 1 (d) toafford p-nitrobenzyl 3,5-bis(tert-butyldimethylsilyloxy)-(α-bromo-2,6-xylate which was subsequentlyreacted with N-[N-(tert-butoxy-carbonyl)-L-seryl]-L-cysteine methylester in an analogous manner to the procedure described in Example 1(f)to yield p-nitrobenzyl 3,5-bis(tert-butyldimethylsilyloxy)-6-[[[(R)-2-[(S)-(1-tertbutoxyformamido)-3-hydroxypropionylamino]-2-(methoxycarbonyl)ethyl]thio]methyl]-2-methylbenzoate.

¹ H-NMR (250 MHz, CDCl₃ ): δ0.27(s,6H); 0.30(s,3H); 0.31(s,3H);0.99(s,9H); 1.01(s,9H); 1.44(s,9H); 2.02(s,3H); 2.87(dd, J=14 Hz and 6Hz, 1H); 2.95 (dd, J=14 Hz and 5 Hz, 1H); 3.04 (m, 1H); 3.64-3.76 (m,1H) superimposed by 3.70(s,3H), and by 3.74(d,J=12 Hz,1H); 3.79(d,J=12Hz, 1H); 4.10(m, 1H); 4.27(broad s,1H); 4.71(m, 1H); 5.50(s,2H); 5.63(broad d, J=8 Hz, 1H); 6.36 (s, 1H); 7.12 (broad d, J=8 Hz, 1H); 7.66(d,J=8 Hz,2H); 8.24(d,J=8 Hz,2H) ppm

(f) A mixture of 4.78 g of p-nitrobenzyl3,5-bis(tert-butyldimethylsilyloxy)-6-[[[(R)-2-[(S)-(1-tert-butoxyformamido)-3-hydroxypropionylamino]-2-(methoxycarbonyl)ethyl]thio]methyl]-2-methylbenzoateand 1.43 g of 5% palladium on charcoal in 100 ml of ethyl acetate washydrogenated for 1 hour at atmospheric pressure. The mixture wasfiltered, and the filtrate was washed successively with 50 ml of 1Nhydrochloric acid and with 100 ml of brine. The organic layer was driedover sodium sulfate and evaporated in vacuo to afford 3.98 g of crude3,5-bis(tert-butyldimethylsilyloxy)-6-[[[(R)-2-[(S)-1-tert-butoxyformamido)-3-hydroxypropionylamino]-2-(methoxycarbonyl)ethyl]thio]methyl]-2-methylbenzoicacid.

¹ H-NMR (250 MHz, CDCl₃): δ0.20(s,6H); 0.25(s,6H); 1.00(s,9H); 1.02 (s,9H); 1.50 (s, 9H); 2.17 (s, 3H); 2.90-3.15 (m, 2H); 3.6-4.9 (m, ˜6H)superimposed by 3.77(s,3H); 6.43 (s, 3H); 7.58 (d, J=8 Hz, 1H) ppm

EXAMPLE 14

To a solution of 61 mg of methyl (4R,7S)-7-amino-12,14-bis(tert-butyldimethylsilyloxy)-1,3,4,5,6,7,8,10-octahydro-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylateand 21 mg N-(tert-butoxycarbonyl)-L-serine in 2 ml of acetonitrile,cooled to 0° C., were added 20 mg ofN-(dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride. The mixturewas stirred at 0° C. for 4 hours, then diluted with 30 ml of ethylacetate, and washed successively with 0.5N hydrochloric acid, water, 5%sodium bicarbonate solution, and with brine. The organic layer was driedover sodium sulfate and evaporated in vacuo. The residue was dissolvedin 2 ml of methanol, 20 mg of ammonium fluoride were added, and themixture was stirred for 30 minutes at room temperature. The mixture wasdiluted with 40 ml of ethyl acetate and washed with 30 ml of water. Theorganic layer was dried over sodium sulfate, and the solvent wasevaporated in vacuo. The solid residue was triturated with 2 ml ofether, then hexane was added, and the white solid was isolated byfiltration to give 22 mg of methyl (4R,7S)-7-[(S)-2-(1-tert-butoxyformamido)-3-hydroxypropionylamino]-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylate.

¹ H-NMR (250 MHz, DMSO-d₆): δ1.36(s,9H); 1.90(s,3H); 2.72(dd, J=14 Hzand 10 Hz, 1H); 3.08 (dd, J=14 Hz and 4 Hz, 1H); 3.44-3.72 (m, 2H)superimposed by 3.49 (d,J=10 Hz, 1H), and by 3.61(s,3H); 3.80(d,J=10 Hz,1H); 4.06-4.28 (m, 2H); 4.52(m, 1H); 4.64(m, 1H); 5.25(t,J=5 Hz, 1H);6.46(s, 1H); 6.83(d,J=8 Hz, 1H); 8.31(d,J=7 Hz,1H); 8.37 (d,J=9 Hz, 1H);9.51 (s, 1H); 9.53 (s, 1H) ppm

The starting material used above was prepared as follows:

(a) To 15 ml of trifluoroacetic acid, cooled to 0° C., were added 1.07 gof tert-butyl (4R, 7S)-12,14-bis(tert-butyldimethylsilyloxy)-1,3,4,5,6,7,8,10-octahydro-4-methoxycarbonyl11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7carbamate.The solution was stirred at 0° C. for 30 minutes, and then the solventwas evaporated in vacuo. The residue was dissolved in 50 ml of ethylacetate, and the solution was washed successively with saturated sodiumcarbonate solution and with brine. The organic layer was dried oversodium sulfate and evaporated in vacuo. Crystallization of the residualmaterial from hexane at -20° C. yielded 0.71 g of methyl (4R,7S)-7-amino-12,14-bis-(tert-butyldimethylsilyloxy)-1,3,4,5,6,7,8,10-octahydro-11-methyl-6,10-dioxo-9,2,5-benzooxathiaazacyclododecine-4-carboxylateas white crystals of m.p. 135°-141° C. (dec.).

EXAMPLE 15

tert-Butyl (S)-12,14-bis(tert-butyldimethylsilyloxy)-1,3,4,5,6,7,8,10-octahydro-11-methyl-6,10-dioxo-9,2,5-benzooxathiaazacyclododecine-7-carbamatewas subjected in an analogous manner to the procedures described inExample 14 to give tert-butyl[(S)-2-hydroxy-1-[[(S)-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-11-methyl-6,10-dioxo-9,2,5-benzooxathiaazacyclododecin-7-yl]carbamoyl]ethyl]carbamate.

¹ H-NMR (250 MHz, DMSO-d₆): δ1.37(s,9H); 1.89(s,3H); 2.54(m, 1H);2.74(m, 1H); 3.13(m, 1H); 3.33(s,3H); 3.48-3.73(m, 4H); 4.09(m, 1H);4.28(m, 1H); 4.94 (t, J=5 Hz, 1H); 6.44 (s, 1H); 6.84 (d, J=8 Hz, 1H);8.06 (d, J=7 Hz, 1H); 8.25(m, 1H); 9.45(s,1H); 9.48(s,1H) ppm

The starting material used above was prepared as follows:

(a) tert-Butyl(S)-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamatewas subjected in an analogous manner to the procedure described inExample 13 (d) to yield, after chromatographic purification, tert-butyl(S)-12,14-bis(tertbutyldimethylsilyloxy)-1,3,4,5,6,7,8,10-octahydro-11-methyl6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamateas a white-off solid.

EXAMPLE 16

(R)-3,5-bis(tert-butyldimethylsilyloxy)-2-[[[2-(3-hydroxypropionylamido)-2-(methoxycarbonyl)ethyl]thio]methyl]-6-methyl-benzoatewas cyclized and the protection groups subsequently cleaved off in ananalogous manner to the procedure described in Example 13 to yield,after chromatographic purification and cristallization from ethylacetate/hexane, methyl(R)-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylateas white solid.

¹ H-NMR (400 MHz,DMSO-d₆): δ1.86(s,3H); 2.44(m, 1H); 2.59(dd, J=14 Hzand 11 Hz, 1H); 2.91 (m, 1H); 3.05(dd, J=14 Hz and 4 Hz, 1H);3.65(s,3H); 3.67 (d, J=10 Hz, 1H); 3.71 (d, J=10 Hz, 1H); 4.51 (m, 1H);4.59-4.70 (m, 2H); 6.42(s,1H); 8.55(d,J=8 Hz, 1H); 9.45(s, 1H); 9.49(s,1H) ppm

The starting material used above was prepared as follows:

(a) 2,2,2-Trichloroethyl 3,5-dihydroxy-2,6-dimethylbenzoate wassubjected in an analogous manner to the procedure described in Example13(d) to yield 2,2,2-trichloroethyl 3,5-bis(tert-butyldimethylsilyloxy)-2, 6-dimethylbenzoate, m.p. 90°-92° C.

(b) 2,2,2-Trichloroethyl 3,5-bis(tert-butyldimethylsilyloxy)-2,6-dimethylbenzoate was subjected in ananalogous manner to the procedure described in Example 1(d) to providecrude 2,2,2-trichloroethyl 2-bromomethyl-3,5-bis(tert-butyldimethylsilyloxy)-6-methylbenzoate which was used withoutfurther purification.

(c) To a solution of 76 g of 1,3-propanediol in 0.5 1 of pyridine,cooled to 0° C., were added 278 g of trityl chloride. The mixture wasstirred at room temperature for 18 hours, and then the solvent wasevaporated in vacuo. The residue was taken up in 600 ml of ethylacetate. Unsoluble material was filtered off, and the filtrate waswashed with 100 ml portions of 3N hydrochloric acid, water, 5% sodiumbicarbonate solution and brine. The organic layer was dried over sodiumsulfate and evaporated in vacuo. Crystallization of the residualmaterial from 0.5 1 of ethyl acetate afforded 167.3 g of3-trityloxy-1-propanol as white crystals of m.p. 117°-118° C.

(d) To a solution of 91.4 g of oxalyl chloride in 1.12 1 of methylenechloride, pre-cooled to -70° C., a solution of 123.4 g ofdimethylsulfoxide in 0.18 l of methylene chloride was added within 25minutes. The solution was stirred for 10 minutes at -70° C. Then, asolution of 143.3 g of 3-trityloxy-1-propanol in 0.6 l of methylenechloride was added within 25 minutes, the temperature being maintainedat -65° to -70° C. Stirring was continued for another 10 minutes, andthen, 182 g of triethylamine were added within 5 minutes at -65° to -70°C. The mixture was stirred for 20 minutes at -70° C. and then allowed towarm up to 10° C. within 1 hour. After the addition of 1.2 1 of water,stirring was continued for 5 minutes. The layers were separated, and theaqueous phase was extracted with 1.2 l of methylene chloride. Theorganic phases were washed with 1.2 l of water, dried over sodiumsulfate, and the solvent was evaporated in vacuo. The residue wascrystallized from ethyl acetate/hexane to yield 116.0 g of3-trityloxypropionaldehyde as white crystals of m.p. 97°-98° C.

(e) To a stirred solution of 41.1 g of 3-trityloxy-propionaldehyde in330 ml of acetone and 120 ml of water were added portionwise, within 90minutes, 20.65 g of potassium permanganate, the temperature of themixture being maintained at 20° to 25° C. Stirring was continued for 90minutes, and then the pH of the mixture was set to 5 by addition of 13ml of 3N hydrochloric acid. Within 40 minutes, there were added dropwise200 ml of 38% sodium bisulfite solution at a temperature of 20° to 25°C. The pH was lowered to 2 by the addition of 60 ml of 3N hydrochloricacid, and subsequently the mixture was extracted with 11 of ethylacetate. The organic layer was washed with water, dried over sodiumsulfate, and the solvent was evaporated in vacuo. The residue wascrystallized from ethyl acetate/hexane to yield 35.5 g of3-trityloxy-propionic acid as white solid of m.p. 159°-161° C.

(f) L-Cysteine methyl ester was acylated with 3-trityloxypropionic acidusing in an analogous manner the procedure described in Example 1(e) toyield, after chromatographic purification and cristallization frommethylene chloride/hexane, methyl(R)-2-(3-trityloxy-propionylamino)-3-mercaptopropionate as whitecrystals of m.p. 80°-85° C.

(g) Crude 2,2,2-trichloroethyl 2-bromomethyl-3,5-bis(tert-butyldimethylsilyloxy)-6-methylbenzoate, as obtained from step 16(b) above, was reacted with methyl(R)-2-(3-trityloxypropionylamino)-3-mercapto-propionate in an analogousmanner to the procedure described in Example 1(f) to yield2,2,2-trichloroethyl (R)-3,5-bis(tert-butyldimethylsilyloxy)-6-methyl-2-[[[2-(3-trityloxypropionylamino)-2-methoxycarbonylethyl]thio]methyl]-benzoate.

(h) A mixture of 6.85 g of 2,2,2-trichloroethyl (R)-3,5-bis(tert-butyldimethylsilyloxy)-6-methyl-2-[[[2-(3-trityloxypropionylamino)-2-(methoxycarbonyl)ethyl]thio]methyl]benzoate and 0.11 g of p-toluenesulfonic acid in 115 ml ofmethanol was stirred at 60° C. for 30 minutes. The mixture was cooledand the solvent was evaporated in vacuo. Chromatography of the residueon silica gel using ethyl acetate/methylene chloride/hexane (1:1:1,v/v/v) as eluent afforded 3.97 g of 2,2,2-trichloroethyl(R)-3,5-bis(tert-butyldimethylsilyloxy)2-[[[2-(3-hydroxypropionylamino)-2-(methoxycarbonyl)ethyl]thio]methyl]-6-methyl-benzoateas white-off solid.

(i) 2,2,2-Trichloroethyl(R)-3,5-bis(tert-butyldimethylsilyloxy)-2-[[[2-(3-hydroxypropionylamino)-2-(methoxycarbonyl)ethyl]thio]methyl]-6-methylbenzoate was subjected in an analogous mannerto the procedure described in Example 1 (g) to yield crude (R)-3, 5-bis(tert-butyldimethylsilyloxy)-2-[[[2-(3-hydroxypropionylamino)-2-(methoxycarbonyl)ethyl]thio]methyl]-6-methylbenzoic acid.

EXAMPLE 17

To a solution of 1.30 g of3,5-bis(tert-butyldimethylsilyloxy)-6-[[[(R)-2-[(R)-(1-tert-butoxyformamido)-3-hydroxypropionylamino]-2-(methoxycarbonyl)ethyl]thio]methyl]-2-methylbenzoicacid and 0.69 g of triphenylphosphine in 42 ml of toluene, cooled to 0°C., was added within 1 hour a solution of 0.44 g of diethylazodicarboxylate in 17 ml of toluene. The mixture was stirred foranother 4 hours at 0° C., and then the solvent was evaporated in vacuo.The residue was taken up in ethyl acetate. Upon cooling in an ice-bathand addition of hexane, crystallization of triphenylphosphine-oxide anddiethyl N,N'-hydrazinedicarboxylate occured. The mixture was filteredand the filtrate was evaporated in vacuo. The residue waschromatographed on silica gel using ethyl acetate/hexane (1: 1, v/v) aseluent to yield 0.36 g of tert-butyl (4R, 7R)-12, 14-bis(tert-butyldimethylsilyloxy)-1,3,4,5,6,7,8,10-octahydro-4-methoxycarbonyl-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamate.To a solution of 71 mg of this material in 2 ml of methanol were added20 mg of ammonium fluoride. The mixture was stirred for 1 hour at roomtemperature, then diluted with 30 ml of ethyl acetate and subsequentlywashed with 2 times 30 ml of water. The organic layer was dried oversodium sulfate, and the solvent was evaporated in vacuo. The residue waschromatographed on silica gel using ethyl acetate/hexane (2:1, v/v) aseluent, and the purified product was crystallized from ethylacetate/hexane to afford 22 mg of tert-butyl(4R,7R)-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-4-methoxycarbonyl-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamateas a white solid.

[α]_(D) =-62.4° (c=0.7, EtOAc)

The starting material used above was prepared as follows:

(a) L-Cysteine methyl ester hydrochloride was acylated withN-(tert-butoxycarbonyl)-D-serine by using in an analogous manner theprocedure described in Example 1(e) to yieldN-[N-(tert-butoxycarbonyl)-D-seryl]-L-cysteine methyl ester as whitecrystals of m.p. 105°-106° C.

(b) p-Nitrobenzyl 3,5-bis(tert-butyldimethylsilyloxy)-alphabromo-2,6-xylate was reacted withN-[N-(tert-butoxycarbonyl)-D-seryl]-L-cysteine methyl ester using in ananalogous manner the procedure described in Example 1(f), and theresulting product was subjected in an analogous manner to the proceduredescribed in Example 13 (f) to yield3,5-bis(tert-butyldimethylsilyloxy)-6-[[[(R)-2-[(R)-(1-tert-butoxyformamido)-3-hydroxypropionylamino]-2-(methoxycarbonyl)ethyl]thio]methyl]-2-methylbenzoate.

EXAMPLE 18

2,2,2-Trichloroethyl 3,5-bis(tert-butyldimethylsilyloxy)-2-methylbenzoate was subjected in ananalogous manner to the reaction sequence described in Example 1(d,f,g),and the resulting product was cyclized in an analogous manner to theprocedure described in Example 21 to yield tert-butyl (4R, 7S)-12,14-bis(tert-butyldimethylsilyloxy)-1,3,4,5,6,7,8,10-octahydro-4-methoxycarbonyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamateas a white solid. This material was treated with ammonium fluoride inmethanol in an analogous manner to the procedure described in Example 21to yield, after crystallization from ethyl acetate/hexane, tert-butyl(4R,7S)-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-4-methoxycarbonyl-6,10-dioxo-9,2,5-benzoxathiaazacyclo-dodecine-carbamateas a white solid.

¹ H-NMR (250 MHz, DMSO-d₆): δ1.42 (s, 9H); 2.87 (dd, J=14 Hz and 11 Hz,1H); 3.05 (dd, J=14 Hz and 4 Hz, 1H); 3.64 (s, 3H); 3.85 (d, J=10 Hz,1H); 4.17-4.45 (m, 3H); 4.59-4.83(m,2H); 6.50(d,J=2.5 Hz, 1H);6.71(d,J=2.5 Hz, 1H); 7.44 (d,J=8 Hz,1H); 8.29(d,J=8 Hz, 1H);9.61(s,1H); 9.80(s, 1H) ppm

The starting material used above was prepared as follows:

(a) To a solution of 57.0 g of 3,5-dihydroxy-2-methylbenzoic acid in 0.5l of dimethylformamide were added 125 g of imidazole and 169 g oftert-butyldimethylchlorosilane, and the mixture was stirred for 24 hoursat 60° C. After cooling, the mixture was poured into 2 l of ice-waterand subsequently extracted twice with 2.5 l portions of hexane. Theorganic layer was washed with 2 l of 5% sodium bicarbonate solution andtwice with 2 l of ice-cold water. The organic layer was dried oversodium sulfate, and the solvent was evaporated in vacuo. The residualoil was crystallized from methanol to give 130 g oftert-butyldimethylsilyl3,5-bis(tert-butyldimethyl-silyloxy)-2-methylbenzoate as white crystalsof m.p. 64° C.

(b) To a solution of 110 g of tert-butyldimethylsilyl3,5-bis(tert-butyldimethylsilylyloxy)-2-methylbenzoate in 1 l oftetrahydrofuran were added 1.5 l of 65% aqueous acetic acid, and themixture was stirred at room temperature for 2 hours. The solvent wasevaporated in vacuo, the residue was taken up in 0.5 l of toluene, andthe solvent was evaporated again in vacuo to give 85 g of crude3,5-bis(tert-butyldimethylsilyloxy)-2-methylbenzoic acid. To a solutionof this material in 0.5 l of methylene choride were added 36.0 g of2,2,2-trichloroethanol and 0.3 g of 4-dimethylaminopyridine. Thesolution was cooled to 5° C., and then, a solution of 60 g ofdicyclohexylcarbodiimide in 250 ml of methylene chloride was addeddropwise within 45 minutes. The mixture was stirred for 1 hour, thetemperature being allowed to raise to room temperature. The precipitateddicyclohexylurea was removed by filtration, the filtrate was evaporatedin vacuo, and the residue was chromatographed on silicagel usinghexane/ethyl acetate (50:1, v/v) as eluent to yield 96 g of2,2,2-trichloroethyl 3,5-bis(tert-butyldimethylsilyloxy)-2-methyl-benzoate as an oil.

¹ H-NMR (250 MHz, CDCl₃): δ0.20(s,3H); 0.22(s,3H); 0.98(s,9H);1.02(s,9H); 2.39(s,3H); 4.92(s,2H); 6.53(d,J=2.5 Hz, 1H); 7.15(d,J=2.5Hz, 1H) ppm

EXAMPLE 19

tert-Butyl (4R, 7S)-12,14-bis(tert-butyldimethylsilyloxy)-1,3,4,5,6,7,8,10-octahydro-4-methoxycarbonyl-11-propyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamatewas subjected in an analogous manner to the procedures described inExample 14 to yield methyl (4R,7S)-7-[(S)-2-(1-tert-butoxyformamido)-3-hydroxypropionylamino]-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-11-propyl-6,10-dioxo-9,2,5-benzooxathiaazacyclododecine-4-carboxylate.

¹ H-NMR (250 MHz, DMSO-d₆): δ0.84 (t,J=5 Hz ,3H); 1.32-1.50 (m, 2H)superimposed by 1.36 (s, 9H); 2.16-2. 44 (m, 2H); 2.68 (dd, J=14 Hz and11 Hz, 1H); 3.04 (dd, J=14 Hz and 4 Hz, 1H); 3.44-3.68(m,4H);3.61(s,3H); 3.75 (d, J=10 Hz, 1H); 4.07(dd, J=12 Hz and 2 Hz, 1H);4.19(m, 1H); 4.48 (m, 1H); 4.62(m, 1H); 5.24(dd, J=12 Hz and 2 Hz, 1H);5.26 (t,J=5 Hz, 1H); 6.45(s, 1H); 6.82(d,J-8 Hz, 1H); 8.26(d,J=8 Hz,1H); 8.37(d,J=9 Hz, 1H); 9.46(s,1H); 9,52(s, 1H) ppm

The starting material used above was prepared as follows:

(a) To 168.2 g of 3,5-dihydroxy-2-methylbenzoic acid in 0.6 l ofmethanol were added 10 ml of 98% sulfuric acid, and the mixture washeated at reflux for 18 hours. The solution was cooled to roomtemperature and neutralized by the addition of about 30 ml of 28% sodiumhydroxide solution. The solvent was evaporated in vacuo, and the residuewas taken up in 0.7 l of ethyl acetate. The solution was washedsuccessively with saturated sodium carbonate solution and brine, driedover sodium sulfate, and the solvent was evaporated in vacuo. Theresidue was crystallized from ethyl acetate/hexane to yield 162.9 g ofmethyl 3,5-dihydroxy-2-methylbenzoate, m.p. 136°-138° C.

(b) To a solution of 27.3 g of methyl 3,5-dihydroxy-2-methylbenzoate and21.8 g of allyl bromide in 300 ml of dimethylformamide were addedportionwise, within 30 minutes, 8.64 g of a 55% dispersion of sodiumhydride in mineral oil, the temperature being held between 10° and 20°C. The mixture was stirred at room temperature for 3 hours, and thenpoured into ice-water. The pH was adjusted to about 3 by the addition of3N hydrochloric acid, and the mixture was extracted with 600 ml of ethylacetate. The organic layer was washed 5 times with 100 ml of water,dried over sodium sulfate, and the solvent was evaporated in vacuo. Theresidue was chromatographed on silica gel using ethyl acetate/methylenechloride/hexane (1:1:3, v/v/v) as eluent, and the purified products werecrystallized from ethyl acetate/hexane to afford 4.1 g of methyl3-allyloxy-5-hydroxy-2-methylbenzoate, m.p. 86°-88° C., and 5.61 g ofmethyl 5-allyloxy-3-hydroxy-2-methylbenzoate, m.p. 84°-87° C.

(c) Under an argon atmosphere, 3.74 g of methyl3-allyloxy-5-hydroxy-2-methylbenzoate were heated to 180° C. for 1.5hours The crude product was chromatographed on silica gel usingacetone/hexane (1:4, v/v) as eluent, and the major product wascrystallized from ethyl acetate/hexane to afford 2.76 g of methyl2-allyl-3,5-dihydroxy-6-methylbenzoate as white crystals of m.p. 72°-95°C.

(d) A mixture of 4.67 g of methyl 2-allyl-3,5-dihydroxy-6-methylbenzoateand 0.23 g of 5% palladium on charcoal in 40 ml of ethyl acetate washydrogenated for 2 hours at atmospheric pressure. The mixture wasfiltered and the filtrate evaporated in vacuo. Crystallization of theresidue from ethyl acetate/hexane afforded 4.30 g of methyl3,5-dihydroxy-2-methyl-6-propylbenzoate, m.p. 88°-91° C.

(e) To a solution of 4.61 g of methyl3,5-dihydroxy-2-methyl-6-propylbenzoate in 20 ml of methylene chloridewere added at 0° C. within 30 minutes 50 ml of a 1M solution of borontribromide in methylene chloride. The mixture was stirred for 5 hours atroom temperature and then diluted with 100 ml of ethyl acetate. Themixture was washed with water and with brine, and the organic layer wasdried over sodium sulfate and evaporated in vacuo to yield crude3,5-dihydroxy-2-methyl-6-propylbenzoic acid.

¹ H-NMR (250 MHz, DMSO-d₆): δ0.85 (t,J=7 Hz,3H); 1.36-1.50(m,2H);1.91(s,3H); 2.30-2.40 (m, 2H); 6.34(s, 1H); 9.05(s, 1H); 9.13(s, 1H);12.70(broad s, 1H) ppm

(f) A mixture of 5.0 g of 3,5-dihydroxy-2-methyl-6-propyl benzoic acid,4.48 g of 2,2,2-trichloroethanol, and 0, 1 ml of 98% sulfuric acid in 40ml of toluene was heated at reflux for 3 hours, water being constantlyremoved by a Dean-Stark trap. The mixture was cooled, diluted with 80 mlof ethyl acetate and washed with brine. The organic layer was dried oversodium sulfate, and the solvent was evaporated in vacuo. Chromatographyof the residue on silica gel using acetone/hexane (1:6, v/v) as eluentafforded 7.1 g of 2,2,2-trichloroethyl3,5-dihydroxy-2-methyl-6-propylbenzoate as an oil.

¹ H-NMR (90 MHz, CDCl₃): δ0.90 (t, 7 Hz, 3H); 1.3-1.7 (m, 2H); 2.13 (s,3H); 2.3-2.6 (m, 2H); 5.00 (s,2H); 5.70(s,1H); 5.80(s, 1H); 6.33(s, 1H)ppm

(g) 2,2, 2-Trichloroethyl 3,5-dihydroxy-2-methyl-6-propylbenzoate wassubjected in an analogous manner to a reaction sequence described inExample 13 (d) and in Example 1 (d, f, g), and the resulting product wascyclized in an analogous manner to the procedure described in Example 1to yield tert-butyl (4R, 7S)-12,14-bis(tert-butyldimethylsilyloxy)-1,3,4,5,6,7,8,10-octahydro-4-methoxycarbonyl-6,10-dioxo-11-propyl-9,2,5-benzoxathiaazacyclododecine-7-carbamate

EXAMPLE 20

tert-Butyl(4R,7S)-12,14-bis(tert-butyldimethylsilyloxy)-1,3,4,5,6,7,8,10-octahydro-4-methoxycarbonyl-11-methyl-6,10-dioxo-13-propyl-9,2,5-benzoxathiaazacyclododecine-7-carbamatewas treated with ammonium fluoride in methanol in an analogous manner tothe procedure described in Example 13 to yield tert-butyl(4R,7S)-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-4-methoxycarbonyl-11-methyl-6,10-dioxo-13-propyl-9,2,5-benzoxathiaazacyclododecine-7-carbamateas a white solid.

¹ H-NMR (250 MHz, DMSO-d₆): δ0.89(t,J=6 Hz, 1H); 1.32-1.52(m, 2H)superimposed by 1.41 (s, 9H); 1.97 (s, 3H), 2.58 (m, 2H); 2.88 (dd, J-14Hz and 10 Hz, 1H); 3.06 (dd, J=14 Hz and 4 Hz, 1H); 3.48 (d, J=11 Hz,1H); 3.63 (s, 3H); 3.89 (d,J=l1 Hz, 1H); 4.21(dd, J=12 Hz and 2 Hz, 1H);4.36(m, 1H); 4.56 (m, 1H); 4.89 (dd, J=12 Hz and 2 Hz, 1H); 7.23 (d, J=8Hz, 1H); 8.19 (d,J=8 Hz, 1H); 8.27 (s, 1H); 8.29(s, 1H) ppm

The starting material used above was prepared as follows:

(a) To a solution of 18.22 g of 3,5-dihydroxy-2,6-dimethylbenzoic acidand 24.2 g of allyl bromide in 200 ml of dimethylformamide were addedportionwise within 30 minutes 8.70 g of a 55% dispersion of sodiumhydride in mineral oil, the temperature being held between 20° and 30°C. The mixture was stirred at 20° C. for 3 hours, then poured into 400ml of ice-water, and the mixture was extracted with 400 ml of ethylacetate. The organic layer was washed 4 times with 200 ml of water,dried over sodium sulfate, and the solvent was evaporated in vacuo. Theresidue was chromatographed on silica gel using acetone/hexane (1:6,v/v) as eluent to give 5.34 g of allyl3-allyloxy-5-hydroxy-2,6-dimethylbenzoate as a coulourless oil.

¹ H-NMR (250 MHz,CDCl₃): δ2.10(s,6H); 4.43-4.47(m, 2H); 4.70-4.90(m,3H); 5.24-5.46 (m, 4H); 5.95-6.10 (m, 2H); 6.38 (s, 1H) ppm

(b) Under an argon atmosphere, 5.06 g of allyl3-allyloxy-5-hydroxy-2,6-dimethylbenzoate were heated to 185° C. for 2.5hours to give 5.02 g of allyl 4-allyl-3,5-dihydroxy-2,6-dimethylbenzoateas an oil.

¹ H-NMR (250 MHz, CDCl₃): δ2.11(s,6H); 3.46-3.50(m, 2H); 4.80-4.85(m,2H); 4.94 (broad s,2H); 5.13-5.46(m, 4H); 5.88-6.11(m,2H) ppm

(c) A solution of 5.00 g of allyl4-allyl-3,5-dihydroxy-2,6-dimethylbenzoate in 80 ml of ethyl acetate wascooled to 0° C., and then treated with 90 mg of palladium acetate and332 mg of triethyl phosphite. After stirring for 5 minutes, 2.04 g ofN-methylpyrrolidine were added, a precipitate being formed immediately.Stirring was continued at 0° C., and then, the mixture was partitionedbetween ethyl acetate and 1N hydrochloric acid. The organic layer waswashed with brine, dried over sodium sulfate, and the solvent wasevaporated in vacuo. The residual oil was dissolved in 50 ml of ethylacetate, and the solution was hydrogenated at atmospheric pressure for 1hour in the presence of 100 mg of 5% palladium on charcoal. The catalystwas filtered off, and the solvent was evaporated in vacuo to yield 3.36g of 3,5-dihydroxy-2,6-dimethyl-4-propylbenzoic acid as a coulourlessoil.

¹ H-NMR (250 MHz, DMSO-d₆): δ0.90(t,J=7 Hz,3H); 1.35-1.50(m,2H);2.00(s,6H); 2.52-2.59 (m, 2H); 7.96 (s, 2H); 12.85 (broad s, 1H) ppm

(d) 3,5-Dihydroxy-2,6-dimethyl-4-propylbenzoic acid was subjected in ananalogous manner to the procedure described in Example 19 (f) to yield2,2,2-trichloroethyl 3,5-dihydroxy-2,6-dimethyl-4-propylbenzoate as anoil.

(e) 2,2,2-Trichloroethyl 3,5-dihydroxy-2,6-dimethyl-4-propylbenzoate wassubjected in an analogous manner to a reaction sequence described inExample 13 (d) and in Example 1 (d, f,g), and the resulting product wascyclized in an analogous manner to the procedure described in Example 1to yield tert-butyl (4R, 7S)-12,14-bis(tert-butyldimethylsilyloxy)-1,3,4,5,6,7,8,10-octahydro-4-methoxycarbonyl-11-methyl-6,10-dioxo-13-propyl-9,2,5-benzoxathiaazacyclododecine-7-carbamate.

EXAMPLE 21

To a solution of 36.4 g of 6-[[[(R)-2-[(S)-2-(1-tert-butoxyformamido)-3-hydroxypropionylamino]-2-(methoxycarbonyl)ethyl]thio]methyl]-3-(tert-butyldimethylsilyloxy)-5-methoxy-2-methylbenzoicacid and 22.3 g of triphenylphosphine in 1.4 l of toluene was added at0° C. within 10 minutes a solution of 15.1 g of diethyl azodicarboxylatein 300 ml of toluene. The mixture was stirred for 5 hours at 0° C., andthen the solvent was evaporated in vacuo. To the residue were added 300ml of methylene chloride, and the mixture was stirred at 0° C. for 15minutes. The precipitate was filtered off, and the filtrate wasevaporated in vacuo. Chromatography of the residue on silica gel usingethyl acetate/hexane (2:3, v/v) as eluent and crystallization of thepurified product from ethyl acetate/hexane provided 19.4 g of tert-butyl(4R,7S)-14-(tertbutyldimethylsilyloxy)-1,3,4,5,6,7,8,10-octahydro-12-methoxy-4-methoxycarbonyl-11-methyl-6,10,-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamateas white crystals of m.p. 157°-158° C. This material was treated withammonium fluoride in methanol in an analogous manner to the proceduredescribed in Example 13 to yield, after crystallization from ethylacetate/hexane tert-butyl (4R,7S)-1,3,4,5,6,7,8,10-octahydro-14-hydroxy-12-methoxy-4-methoxycarbonyl-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamateas a white solid.

¹ H-NMR (400 MHz,DMSO-d₆): δ1.41 (s, 9H); 1.94 (s, 3H); 2.90 (dd, J=14Hz and 10 Hz, 1H); 3.04 (dd, J=14 Hz and 4 Hz, 1H); 3.40 (d, J=l1 Hz,1H); 3.63 (s, 3H); 3.71(s,3H); 3.81(d,J=l1 Hz, 1H); 4.22(m, 1H); 4.35(m,1H); 4.86 (m, 1H); 6.53 (s, 1H); 7.27 (d, J=8 Hz, 1H); 8.15 (d, J=8 Hz,1H); 9.73 (s, 1H) ppm

The starting material used above was prepared as follows:

(a) To a solution of 31.7 g of p-nitrobenzyl3,5-dihydroxy-2,6-dimethylbenzoate in 200 ml of acetone were added 16.6g of potassium carbonate and 17.1 g of methyl iodide, and the mixturewas heated at reflux for 15 hours. The reaction mixture was filtered,and the filter cake was washed with 200 ml of ethyl acetate. Thefiltrate was washed successively with 0.5N hydrochloric acid and withbrine, dried over sodium sulfate, and the solvent was evaporated i nvacuo. The remaining oil was dissolved in 150 ml of dimethylformamide,and the solution was cooled to 0° C. After the addition of 18.1 g oftert-butyldimethylchlorosilane and 13.1 g of triethylamine, the mixturewas stirred at 0° C. for 3 hours. The mixture was diluted with 300 ml ofethyl acetate and subsequently washed 5 times with 10 0 ml of water. Theorganic layer was dried over sodium sulfate, and the solvent wasevaporated in vacuo. The remaining oil was chromatographed on silica gelusing acetone/hexane (1:5, v/v) as eluent to yield, aftercrystallization from ethyl acetate/hexane, 14,5 g of p-nitrobenzyl3,5-bis(tert-butyldimethylsilyloxy)-2,6-dimethylbenzoate and 13.8 g ofp-nitrobenzyl3-(tert-butyldimethylsilyloxy)-5-methoxy-2,6-dimethylbenzoate, m.p.80°-81° C., as the major products.

(b) p-Nitrobenzyl3-(tert-butyldimethylsilyloxy)-5-methoxy-2,6-dimethylbenzoate wassubjected in an analogous manner to the procedures described in Example1 (d, f) and 13 (f) to yield6-[[[(R)-2-[(S)-2-(1-tert-butoxyformamido)-3-hydroxypropionylamino]-2-(methoxycarbonyl)ethyl]thio]methyl]-3-(tert-butyldimethylsilyloxy)-5-methoxy-2-methylbenzoicacid

¹ H-NMR (250Mz, CDCl₃): (inter alia) δ1.47 (broad s, ˜10H); 2.17 (s,3H); 2.90 (dd, 1H); 3.09(dd, 1H); 3.74(s,3H); 3.76(s,3H); 4.17(dd, 1H);4.64(m, 1H); 4.76(m, 1H); 5.73(d, 1H); 6.37(s, 1H); 7.26(s,1H); 7.41(d,1H) ppm

EXAMPLE 22

(R)-5-(tert-Butyldimethylsilyloxy)-2-[[[2-(3-hydroxypropionylamino)-2-(methoxycarbonyl)ethyl]thio]methyl]-5-methoxy-6-methylbenzoicacid was cyclized and the protection groups were subsequently cleavedoff in an analogous manner to the procedure described in Example 13 toyield, after chromatographic purification and cristallization frommethanol/diethylether/hexane, methyl(R)-1,3,4,5,6,7,8,10-octahydro-14-hydroxy-12-methoxy-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylateas a white solid.

¹ H-NMR (250 MHz, DMSO-d₆): δ1.90(s,3H); 2.44(m, 1H); 2.59(dd, 1H);2.90(m, 1H); 3.07 (dd, 1H); 3.65(s,3H); 3.73(s,5H); 4.47-4.73(m,3H);6.50 (s, 1H); 8.55(d, 1H); 9.67(s, 1H) ppm

The starting material used above was prepared as follows:

(a) p-Nitrobenzyl3-(tert-butyldimethylsilyloxy)-5-methoxy-2,6-dimethylbenzoate wassubjected in an analogous manner to the procedures described in Example1 (d), Example 16 (g, h), and in Example 13 (f) to yield(R)-5-(tert-butyldimethylsilyloxy)-[[[2-(3-hydroxypropionylamino)-2-(methoxycarbonyl)ethyl]thio]methyl]-5-methoxy-6-methylbenzoicacid.

EXAMPLE 23

p-Nitrobenzyl3-(tert-butyldimethylsilyloxy)-5-methoxy-2-methyl-6-[[[(R)-2-[(S)-(4-nitrobenzyloxycarbonyloxy)-butyrylamino]-2-(methoxycarbonyl)ethyl]thio]methyl]-benzoic acid was subjected inanalogous manner to the hydrogenation procedure described in Example 13(f), the raw product was cyclized, and subsequently the silyl groupswere cleaved off in an analogous manner to the procedures described inExamples 21 and 13, to yield methyl (4R,8R)-1,3,4,5,6,7,8,10-octahydro-14-hydroxy-12-methoxy-8,11-dimethyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylateas a white solid.

¹ H-NMR (400 MHz, DMSO-d₆): δ1.32(d,J=7 Hz,3H); 1.91(s,3H); 2.48(m, 1H);2.62(dd, J=14 Hz and 11 Hz, 1H); 2.69 (dd, J=14 Hz and 12 Hz, 1H); 3.10(dd, J=14 Hz and 4.5 Hz, 1H); 3.50(d,J=l1 Hz, 1H); 3.65(s,3H);3.72(s,3H); 3.99 (d, J=l1 Hz, 1H); 4.70 (m, 1H); 5.70 (m, 1H); 6.50 (s,1H); 8.45 (d,J=9 Hz, 1H); 9.69(s, 1H) ppm

The starting material used above was prepared as follows:

(a) To a solution of 3.84 g of tert-butyl (S)-3-hydroxybutyrate and 5.86g of 4-dimethylaminopyridine in 20 ml of methylene chloride was added at0° C. within 20 minutes a solution of 6.47 g of p-nitrobenzylchloroformate in 20 ml of methylene chloride. The mixture was stirredfor 3 hours at 0° C., then diluted with 50 ml of methylene chloride andwashed successively with 1N hydrochloric acid, saturated sodiumbicarbonate solution, and with brine. The organic layer was dried oversodium sulfate, and the solvent was evaporated in vacuo. The residualoil was chromatographed on silica gel using ethyl acetate/hexane (1:1,v/v) as eluent to yield 5.10 g of tert-butyl(S)-3-(4-nitrobenzyloxycarbonyloxy)-butyrate as a colourless oil.

(b) A solution of 5.10 g of tert-butyl(S)-3-(4-nitrobenzyloxycarbonyloxy)-butyrate in 20 ml of trifluoroaceticacid was stirred at 0° C. for 90 minutes. The solvent was evaporated invacuo, and the residue was crystallized from methylene chloride/hexaneto yield 3.28 g of (S)-3-(4-nitrobenzyloxycarbonyloxy)-butyric acid aswhite crystals of m.p. 78°-81° C.

(c) To 2.83 g of (S)-3-(4-nitrobenzyloxycarbonyloxy)-butyric acid and1.72 g of N-hydroxy-succinimide in 50 ml of acetonitrile were added 2.88g of N-(dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride. Themixture was stirred at 0° C. for 3 hours. Then, 2.58 g of L-cysteinemethyl ester hydrochloride and 1.52 g of 4-methylmorpholine were added ,and stirring was continued for another 3 hours at 0° C. The solvent wasevaporated in vacuo, the residue was taken up in 150 ml of ethylacetate, and the solution was washed successively with 1N hydrochloricacid, saturated sodium bicarbonate solution, and with saturated sodiumchloride solution. The organic layer was dried over sodium sulfate, andthe solvent was evaporated in vacuo. The remaining oil waschromatographed on silica gel using ethyl acetate/hexane (1:1, v/v) aseluent. Crystallization of the purified product from methylenechloride/hexane provided methyl(R)-2-[(S)-(4-nitrobenzyloxycarbonyloxy)butylamino]-3-mercapto-propionateas a white solid.

¹ H-NMR (250 MHz, CDCl₃): δ1.36 (m, 1H); 1.41 (d, J=6 Hz, 3H); 2.60(ddd, J=14 Hz, 8 Hz and 5 Hz, 2H); 3.01(dd, J=9 Hz and 4 Hz,2H);3.79(s,3H); 4.87(m, 1H); 5.14-5.32(m, 3H); 6.51(d,J=7 Hz, 1H);7.55(d,J=8 Hz,2H); 8.23 (d,J=8 Hz, 2H) ppm

(d) p-Nitrobenzyl3-(tert-butyldimethylsilyloxy)-5-methoxy-2,6-dimethylbenzoate wassubjected in an analogous manner to the procedures described in Example1 (d) to give a product mixture containing p-nitrobenzyl2-bromomethyl-3-(tert-butyldimethyl-silyloxy)-5-methoxy-6-methylbenzoate.

(e) Crude p-nitrobenzyl2-bromomethyl-3-(tert-butyldimethylsilyloxy)-5-methoxy-6-methylbenzoate,as obtained in step (d), was reacted with methyl(R)-2-[(S)-(4-nitrobenzyloxycarbonyloxy)-butyrylamino]-3-mercapto-propionatein an analogous manner to the procedure described in Example 1 (f) toyieldp-nitrobenzyl-3-(tert-butyldimethylsilyloxy)-5-methoxy-2-methyl-6-[[[(R)-2-[(S)-(4-nitrobenzyloxycarbonyloxy)-butyrylamino]-2-(methoxy-carbonyl)ethyl]thio]methyl]-benzoicacid

EXAMPLE 24

Crude p-nitrobenzyl2-bromomethyl-3-(tert-butyldimethylsilyloxy)-5-methoxy-6-methylbenzoate,as obtained in Example 23 (d), was reacted withN-[N-(tert-butoxycarbonyl)-D-seryl]-L-cysteine methyl ester in ananalogous manner to the procedure described in Example 1 (f), and theproduct was subjected in an analogous manner to the sequence ofprocedures used in Example 23 to yield tert-butyl (4R,7R)-1,3,4,5,6,7,8,10-octa-hydro-14-hydroxy-12-methoxy-4-methoxycarbonyl-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamateas a white solid.

¹ H-NMR (250 MHz, DMSO-d₆): δ1.40 (s, 9H); 1.91 (s, 3H); 2.59(dd, J=14Hz and 11 Hz, 1H); 3.05 (dd, J=14 Hz and 4 Hz, 1H); 3.64 (d, J=10 Hz,1H); 3.65 (s, 3H); 3.72 (s,3H); 3.80(d,J=10 Hz, 1H); 4.32-4.50(m, 1H);4.54-4.72(m, 1H); 6.49(s,1H); 7.07(d,J=8 Hz, 1H); 8.79(d,J=8 Hz, 1H);9.70(s, 1H) ppm

EXAMPLE 25

Crude p-nitrobenzyl2-bromomethyl-3-(tert-butyldimethylsilyloxy)-5-methoxy-6-methylbenzoate,as obtained in Example 23(d), was reacted withN-[N-(tert-butoxycarbonyl)-D-threonyl]-L-cysteine methyl ester in ananalogous manner to the procedure described in Example 1 (f), and theproduct was subjected in an analogous manner to a sequence of proceduresdescribed in Example 23 to yield t-butyl (4R,7R,8R)-1,3,4,5,6,7,8,10-octahydro-14-hydroxy-12-methoxy-4-methoxycarbonyl-8,11-dimethyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamateas a white solid.

¹ H-NMR (250 MHz, DMSO-d₆): δ1.26 (d, 3H); 1.39 (s, 9H); 1.92 (s, 3H);2.51 (dd, 1H); 3.05 (dd, 1H); 3.28 (d, 1H); 3.64 (s, 3H); 3.72 (s, 3H);4.03 (d, 1H); 4.30 (dd, 1H); 4.65 (m, 1H); 5.33 (m, 1H); 6.50 (s, 1H);7.23 (d, 1H); 8.68 (d, 1H); 9.69 (s, 1H) ppm

The starting material used above was prepared as follows:

(a) L-Cysteine methyl ester was acylated withN-(tert-butoxycarbonyl)-D-threonine by using in an analogous manner theprocedure described in Example 1(e) to yield after chromatographicpurification and crystallization from diethylether/hexaneN-[N-(tert-butoxycarbonyl)-D-threonyl]-L-cysteine methyl ester as whitecrystals of m.p. 98°-100° C.

EXAMPLE 26

Crude p-nitrobenzyl2-bromomethyl-3-(tert-butyldimethylsilyloxy)-5-methoxy-6-methylbenzoate,as obtained in Example 23(d), was reacted with methyl(R)-2-(2,2-dimethyl-3-trityloxypropionyiamino)-3-mercapto-propionate inan analogous manner to the procedure described in Example 1 (f), and theproduct was subjected in an analogous manner to a sequence of proceduresdescribed in Example 16(h), 13(f) and 21 to yield tert-butyl(R)-1,3,4,5,6,7,8,10-octahydro-14-hydroxy-12-methoxy-4-methoxycarbonyl-7,7,11-trimethyl-6,10-dioxo-9,2,5-benzooxathiaazacyclododecine-7-carbamateas a white solid.

¹ H-NMR (250 MHz, DMSO-d₆): δ1.15(s,3H); 1.24(s,3H); 1.92 (s,3H);2.72(dd, J=14 Hz and 12 Hz, 1H); 4.14 (dd, J=14 Hz and 4.5 Hz, 1H);3.5-3.7 (m, 2H) superimposed by 3.64 (s, 3H); 3.72 (s, 3H); 4.36 (d,J=11 Hz, 1H); 4.44(d,J=11 Hz, 1H); 4.56(m, 1H); 6.49(s,1H); 8.12(d, 8Hz, 1H); 9.70 (s, 1H) ppm

The starting material used above was prepared as follows:

(a) To a solution of 25.0 g of methyl 3-hydroxy-2,2-dimethylpropanoatein 100 ml of pyridine, cooled to 0° C., were added 55.7 g of tritylchloride. The mixture was stirred at room temperature for 20 hours, andthen the solvent was evaporated in vacuo. The residue was taken up in200 ml of ethyl acetate, and the solution was washed successively withwater, 1N hydrochloric acid, water, saturated sodium bicarbonatesolution and brine. The organic layer was dried over sodium sulfate, andthe solvent was evaporated in vacuo. The residual oil was taken up in 50ml of methylene chloride, and to the solution were added slowly, at 0°C., 300 ml of hexane. Trityl alcohol crystallized and was filtered off.The filtrate was evaporated in vacuo to yield 53.5 g of raw methyl2,2-dimethyl-3-trityloxy-propionate.

(b) A solution of 53.5 g of raw methyl2,2-dimethyl-3-trityloxy-propionate and 16.8 g of potassium hydroxide in100 ml of ethylene glycol was heated to 160° C. for 4 hours. The mixturewas cooled, diluted with 400 ml of ethyl acetate, and then washedsuccessively with ice-cold 1N hydrochloric acid and with brine. Theorganic layer was dried over sodium sulfate, and the solvent wasevaporated in vacuo. The residue was triturated with diethylether,unsoluble material was filtered off, and the filtrate was evaporated.Crystallization of the residue from diethylether/hexane provided 7.5 gof 2,2-dimethyl-3-trityloxy-propanoic acid, m.p. 153°-158° C.

¹ H-NMR(90 MHz,CDCl₃): δ1.23 (s, 6H); 3.17(s,2H); 7.2-7.7(m, 15H) ppm

(c) L-Cysteine methyl ester was acylated with2,2-dimethyl-3-trityloxy-propanoic acid in an analogous manner to theprocedure described in Example 1 (e) to yield after chromatographicpurification methyl(R)-2-(2,2-dimethyl-3-trityloxypropionylamino)-3-mercapto-propionate asan oil.

¹ H-NMR(250 MHz,CDCl₃): δ1.20(s,3H); 1.24(s,3H); 2.93(m,2H); 3.16(s,2H);3.68(s,3H); 4.86 (m, 1H); 7.2-7.6 (m, 15H) ppm

EXAMPLE 27

3-(tert-Butyldimethylsilyloxy)-6-[[[(R)-2-(1-tert-butoxyformamido)-5-ethoxy-3-hydroxypropionylamino]-2-(methoxycarbonyl)ethyl]thio]methyl]-2-methylbenzoicacid was subjected in an analogous manner to the cyclization proceduredescribed in Example 21 to yield, after crystallization from hexane,tert-butyl (4R,7S)-14-(tert-butyldimethylsilyloxy)-1,3,4,5,6,7,8,10-octahydro-12-ethoxy-4-methoxycarbonyl-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamateas white crystals. This material was treated with ammonium fluoride inmethanol in an analogous manner to the procedure described in Example 13to yield, after crystallization from ethyl acetate/hexane, tert-butyl(4R,7S)-1,3,4,5,6,7,8,10-octahydro-14-hydroxy-12-ethoxy-4-methoxycarbonyl-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamateas a white solid.

¹ H-NMR(250 MHz, DMSO-d₆): 1.32(t,J=7 Hz,3H); 1.41(s,9H); 1.93(s,3H);2.84(dd, J=14 Hz and 10 Hz, 1H); 3.05 (dd, J=14 Hz and 4 Hz, 1H); 3.45(d, J=10 Hz, 1H); 3.63 (s,3H); 3.84 (d,J=10 Hz, 1H); 3.94 (q,J=7 Hz,2H);4.21(dd, J=11 Hz and 3 Hz,1H); 4.35(m, 1H); 4.53(m, 1H); 4.89(dd, J=11Hz and 2 Hz, 1H); 6.49(s, 1H); 7.20 (d, J=8 Hz, 1H); 8.16 (d, J=8 Hz,1H); 9.68 (s, 1H) ppm

The starting material used above was prepared as follows:

(a) p-Nitrobenzyl 3,5-dihydroxy-2,6-dimethylbenzoate was subjected in ananalogous manner to the procedure described in Example 21(a), usingethyl bromide instead of methyl iodide, to yield after crystallizationfrom hexane, p-nitrobenzyl3-(tert-butyldimethylsilyloxy)-5-ethoxy-2,6-dimethylbenzoate as whitecrystals, m.p.75°-76° C.

(b) p-Nitrobenzyl3-(tert-butyldimethylsilyloxy)-5-ethoxy-2,6-dimethylbenzoate wassubjected in an analogous manner to the procedures described in Example1(d,f) and 13(f) to yield3-(tert-butyldimethylsilyloxy)-6-[[[(R)-2-(1-tert-butoxyformamido)-5-ethoxy-3-hydroxypropionylamino]-2-(methoxycarbonyl)ethyl]thio]methyl]-2-methylbenzoicacid.

EXAMPLE 28

p-Nitrobenzyl2-bromomethyl-3-(tert-butyldimethylsilyloxy)-5-methoxybenzoate wassubjected in an analogous manner to a sequence of procedures describedin Example 1(f) and 13(f), and the product was cyclized in in analogousmanner to the procedure described in Example 21 to yield tert-butyl (4R,7S)-14-(tert-butyldimethylsilyloxy)-1,3,4,5,6,7,8,10-octahydro-12-methoxy-4-methoxycarbony1-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamate as whitecrystals. This material was treated with ammonium fluoride in methanolin an analogous manner to the procedure described in Example 13 toyield, after crystallization from methanol/ethyl acetate/hexane,tert-butyl (4R,7S)-1,3,4,5,6,7,8,10-octahydro-14-hydroxy-12-methoxy-4-methoxycarbonyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamateas a white solid.

¹ H-NMR(250 MHz, DMSO-d₆): δ1.42 (s, 9H); 2.88 (dd, 1H); 3.05 (dd,1H).;3.64 (s,3H); 3.71 (s,3H); 3.86 (d, 1H); 4.20-4.45 (m, 3H);4.55-4.83 (m, 2H); 6.60 (d, 1H); 6.79 (d, 1H); 7.42 (d, 1H); 8.28 (d,1H) ppm

The starting material used above was prepared as follows:

(a) 3,5-Dihydroxy-2-methylbenzoic acid was subjected in an analogousmanner to the procedure described in Example 13(b) to yieldp-nitrobenzyl 3,5-dihydroxy-2-methylbenzoate as pale yellow crystals ofm.p. 160°-163° C.

(b) p-Nitrobenzyl 3,5-dihydroxy-2-methylbenzoate was subjected in ananalogous manner to the procedure described in Example 21 (a) to yield,after chromatographic purification and crystallization from hexane,p-nitrobenzyl 3-(tert-butyldimethylsilyloxy)-5-methoxy-2-methylbenzoateas white crystals of m.p. 83°-85° C.

(c) p-Nitrobenzyl3-(tert-butyldimethylsilyloxy)-5-methoxy-2-methylbenzoate was subjectedin an analogous manner to the procedure described in Example 1(d) toyield, after crystallization from ethyl acetate/hexane, p-nitrobenzyl2-bromomethyl-3-(tert-butyldimethylsilyloxy)-5-methoxybenzoate as whitecrystals of m.p. 82°-84° C.

EXAMPLE 29

p-Nitrobenzyl2-bromomethyl-3-(tert-butyldimethylsilyloxy)-5-methoxybenzoate wasreacted with N-[N-(tert-butoxycarbonyl)-D-threonyl]-L-cysteine methylester in an analogous manner to the procedure described in Example 1(f), and the product was subjected in-an analogous manner to a sequenceof procedures described in Example 13 (f) and in Example 21 to yieldtert-butyl (4R, 7R, 8R)-14-(tert-butyldimethylsilyloxy)-1,3,4,5,6,7,8,10-octahydro-12-methoxy-4-methoxycarbonyl-8-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamateas white crystals. This material was treated with ammonium fluoride inmethanol in an analogous manner to the procedure described in Example 13to yield, after crystallization from methanol/diethylether/hexane,tert-butyl (4R, 7R, 8R)-1,3,4,5,6,7,8,10-octahydro-14-hydroxy-12-methoxy-4-methoxycarbonyl-8-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamateas a white solid.

¹ H-NMR (250 MHz, DMSO-d₆): δ1.21(d,3H); 1.40(s, 9H); 2.61(dd, 1H);2.90(dd, 1H); 3.66(s,3H); 3.71 (s, 3H); 3.82 (d, 1H); 4.41 (dd, 1H);4.60 (d 1H); 4.79 (m, 1H); 5.32 (m, 1H); 6.58 (d, 1H); 6.71 (d, 1H);7,28 (d, 1H); 9.08 (d, 1H); 10.04 (s, 1H) ppm

EXAMPLE 30

p-Nitrobenzyl2-bromomethyl-3-(tert-butyldimethylsilyloxy)-5-methoxybenzoate wasreacted with N-[N-(tert-butoxycarbonyl)-D-seryl]-L-cysteine methyl esterin an analogous manner to the procedure described in Example 1 (f), andthe product was subjected in an analogous manner to a sequence ofprocedures described in Example 13 (f) and,in Example 21 to yieldtert-butyl (4R, 7R)-14-(tert-butyldimethylsilyloxy)-1,3,4,5,6,78,10-octahydro-12-methoxy-4-methoxycarbonyl-6,10-dioxo-9,2,5-benzoxathiaazacyclo-dodecine-7-carbamateas white crystals. This material was treated with ammonium fluoride inmethanol in an analogous manner to the procedure described in Example 13to yield, after crystallization from diethylether/hexane, tert-butyl(4R,7R)-1,3,4,5,6,7,8,10-octahydro-14-hydroxy-12-methoxy-4-methoxycarbonyl-6,10-dioxo-9,2,5-benzoxathiaazacyclo-dodecine-7-carbamateas a white solid.

¹ H-NMR(250 MHz, DMSO-d₆): δ1.39(s,9H); 2.64(dd, J=14 Hz and 11 Hz, 1H);3.06(dd, J=14 Hz and 4 Hz, 1H); 3.67(s,3H); 3.71(s,3H); 3.81(d,J=12 Hz,1H); 4.22 (m, 1H); 4.33 (d, J=12 Hz, 1H); 4.48 (m, 1H); 4.56 (m, 1H);4.73(m, 1H); 6.61(d,J=2 Hz,1H); 6.74(d,J=2 Hz, 1H); 7.14(d,J=8 Hz,1H);9.02 (d, J=9 Hz, 1H); 10.11 (s, 1H) ppm

EXAMPLE 31

p-Nitrobenzyl2-bromomethyl-3-(tert-butyldimethylsilyloxy)-5-methoxybenzoate wassubjected in an analogous manner to a sequence of procedures describedin Example 16(g,h), Example 13(f), and in Example 21 to yield methyl(4R)-14-(tert-butyldimethylsilyloxy)-1,3,4,5,6,7,8,10-octahydro-12-methoxy-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylate.This material was treated with ammonium fluoride in methanol in ananalogous manner to the procedure described in Example 13 to yield,after crystallization from acetone/hexane, methyl(R)-1,3,4,5,6,7,8,10-octahydro-14-hydroxy-12-methoxy-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylateas a white solid.

¹ H-NMR(250 MHz, DMSO-d₆): δ2.50 (m, partially obscured, 1H); 2.65 (dd,1H); 2.81 (m, 1H); 3.06 (dd, 1H); 3.66 (s, 3H); 3.71 (s, 3H); 3.86 (d,1H); 4.35 (m, 2H); 4.74 (m, 2H); 6.60 (d, 1H); 6.74 (d, 1H); 8.74 (d,1H); 10.03 (s, 1H) ppm

EXAMPLE 32

p-Nitrobenzyl2-bromomethyl-3-(tert-butyldimethylsilyloxy)-5-methoxybenzoate wasreacted with methyl(R)-2-[(R)-3-hydroxy-3-methoxycarbonyl-propionylamino]-3-mercapto-propionatein an analogous manner to the procedure described in Example 1(f), andthe product was subjected in an analogous manner to a sequence ofprocedures described in Example 13(f) and in Example 21 to yielddimethyl (4R,8S)-14-(tert-butyldimethylsilyloxy)-1,3,4,5,6,7,8,10-octahydro-12-methoxy-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4,8-dicarboxylateas a white solid. This material was treated with ammonium fluoride inmethanol in an analogous manner to the procedure described in Example 13to yield, after crystallization from ethyl acetate/hexane, dimethyl (4R,8S)-1,3,4,5,6,7,8,10-octahydro-14-hydroxy-12-methoxy-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4,8-dicarboxylateas a white solid.

¹ H-NMR(250M/{z, DMSO-d₆): δ2.62(dd, J=14 Hz and 11 Hz, 1H); 2.75(dd,J=14 Hz and 3 Hz, 1H); 2.90 (dd, J=14 Hz and 12 Hz, 1H); 3.08 (dd, J=14Hz and 4 Hz, 1H); 3.67(s,3H); 3.72(s,3H)3.73(s,3H); 3.91(d,J=10 Hz, 1H);4.53 (d, J=10 Hz, 1H); 4.74 (m, 1H); 5.61 (dd, J=12 Hz and 3 Hz, 1H);6.65 (d, J=2 Hz, 1H); 6.77 (d, J=2 Hz, 1H); 8.93 (d, J=9 Hz, 1H); 10.15(s, 1H) ppm

The starting material used above was prepared as follows:

(a) L-Cysteine methyl ester was acylated with (R)-hydroxy-1,4-butandioicacid 1-methyl ester in an analogous manner to the procedure described inExample 1(e) to yield after chromatographic purification andcrystallization from ethyl acetate/hexane methyl(R)-2-[(R)-3-hydroxy-3-methoxycarbonylpropionylamino]-3-mercapto-propionateas white crystals of m.p. 80°-81° C.

EXAMPLE 33

p-Nitrobenzyl(R)-3-(tert-butyldimethylsilyloxy)-5-methoxy-2-[[[2-(3-trityloxypropionyl-methyl-amino)-2-(methoxycarbonyl)ethyl]thio]methyl]-benzoatewas subjected in an analogous manner to a sequence of proceduresdescribed in Example 16(h) and 13(f), and the product was cyclized andsubsequently the silyl groups were cleaved off in an analogous manner tothe procedures described in Examples 21 and 13, to yield methyl(R)-1,3,4,5,6,7,8,10-octahydro-14-hydroxy-12-methoxy-5-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylateas a white solid.

¹ H-NMR(250 MHz,DMSO-d₆): (inter alia) δ2.62(s,3H); 2.77(dd, J=15 Hz and10 Hz,1H); 3.03 (dd, J=15 Hz and 5 Hz, 1H); 3.50-3.70 (m, 1H); 3.71 (s,6H); 3.79 (d, J=10 Hz, 1H); 4.15 (d, J=10 Hz, 1H); 4.44-4.60 (m, 2H);5.14 (m, 1H); 6.57 (d, J=2 Hz, 1H); 6.73 (d, J=2 Hz, 1H); 10.16 (s, 1H)ppm

The starting material used above was prepared as follows:

(a) A solution of 0.53 g of p-nitrobenzylalpha-bromo-3-(tert-butyldimethylsilyloxy)-5-methoxy-toluate and 0.17 gof L-cysteine methyl ester hydrochloride in 2 ml of methylenechloride/acetonitrile (1:1, v/v) was cooled to 0° C. and then treatedwith 0.20 g of triethylamine, a precipitate being formed immediately.The mixture was stirred for 4 hours at 0° C. Ethyl acetate was added,and the mixture was washed successively with saturated sodiumbicarbonate solution and with brine. The organic layer was dried oversodium sulfate, and the solvent was evaporated in vacuo. The residualoil was chromatographed on silica gel using ethyl acetate/hexane (1:1,v/v) as eluent to yield 0.34 g of p-nitrobenzyl(R)-2-[[[2-amino-2-(methoxycarbonyl)ethyl]thio]methyl]-3-(tert-butyldimethylsilyloxy)-5-methoxybenzoateas an oil.

¹ H-NMR(90 MHz,CDCl₃): δ0.29(s,6H); 1.03(s,9H); 1.87(broad s,2H);2,05(s,3H); 2.66 (dd, J=14 Hz and 8 Hz, 1H); 2.95 (dd, J=14 Hz and 4 Hz,1H); 3.60 (m, 1H); 3.71(s,3H); 3.80(s,3H); 4.20(m, 2H); 5.49(s,2H); 6.63(d, J=2 Hz, 1H); 7.13 (d, J=2 Hz, 1H); 7.70 (d, J=8 Hz, 2H); 8.33 (d,J=8 Hz, 2H) ppm

(b) A solution of 1.90 g of p-nitrobenzyl(R)-2-[[[2-amino-2-methoxycarbonyl-ethyl]thio]methyl]-3-(tert-butyldimethylsilyloxy)-5-methoxybenzoateand 0.91 g of methyl iodide in 5 ml of acetonitrile was stirred at roomtemperature for 3 hours. The solution was diluted with ethyl acetate andwashed successively with saturated sodium bicarbonate solution and withbrine. The organic layer was dried over sodium sulfate, and the solventwas evaporated in vacuo. The residual oil was chromatographed on silicagel using ethyl acetate/hexane (1:1, v/v) as eluent to yield 0.52 g ofp-nitrobenzyl(R)-2-[[[2-methoxycarbonyl-2-(methylamino)ethyl]thio]methyl]-3-(tert-butyldimethylsilyloxy)-5-methoxybenzoateas an oil.

¹ H-NMR(250 MHz, DMSO-d₆): δ0.26(s,6H); 1.00(s,9H); 2.15 (broad s, 3H);2, 60 (m, 2H); 3.56(s,3H); 3.77(s,3H); 4.00(m, 2H); 5.47(s,2H); 6.57(d,J=2 Hz,1H); 7.01 (d,J=2 Hz, 1H); 7.59(d,J=8 Hz,2H); 8.21 (d, J=8 Hz,2H) ppm

(c) To a stirred suspension of 3.32 g of 3-trityloxypropionic acid in 50ml of methanol was added within 15 minutes a solution of 0.56 g ofpotassium hydroxyde in 10 ml of methanol. Stirring was continued for 30minutes, and then the solvent was evaporated in vacuo. The residual foamwas dissolved in 20 ml of methylene chloride, and to the stirredsolution were slowly added 100 ml of diethylether to causecrystallization. There were obtained 3.40 g of potassium3-trityloxypropionate as a white solid.

To a suspension of 3.30 g of potassium 3-trityloxypropionate in 75 ml oftoluene, cooled to 0° C., was added 0.5 ml of pyridine, and subsequently15 ml of oxalyl chloride were added within 10 minutes. The mixture wasstirred for 30 minutes at room temperature and then evaporated in vacuo.The oily residue was taken up in 30 ml of toluene, and the solvent wasevaporated in vacuo. The residue was taken up in 50 ml of methylenechloride and 50 ml of hexane, and the solution was stirred at 0° C.Unsoluble material was filtered off, and the filtrate was evaporated invacuo to yield 2.81 g of 3-trityloxypropionyl chloride as a white solid.

(d) A solution of 0.64 g of p-nitrobenzyl(R)-2-[[[2-(methoxycarbonyl)-2-(methylamino)-ethyl]thio]methyl]-3-(tert-butyldimethylsilyloxy)-5-methoxybenzoateand 0.58 g of 3-trityloxypropionyl chloride in 6 ml of methylenechloride was treated at 0° C. with 0.17 g of triethylamine. The mixturewas stirred at 0° C. for 30 minutes, then diluted with methylenechloride, and washed successively with 5% sodium bicarbonate solutionand brine. The organic layer was dried over sodium sulfate, and thesolvent was evaporated in vacuo. The residual oil was chromatographed onsilica gel using ethyl acetate/hexane (1:2, v/v) as eluent to yield 0.65g of p-nitrobenzyl(R)-3-(tert-butyldimethylsilyloxy)-5-methoxy-2-[[[2-(3-trityloxypropionyl-methylamino)-2-methoxycarbonyl-ethyl]thio]methyl]-benzoateas an oil.

¹ H-NMR(250 MHz, CDCl₃): inter alia, δ0.23(s,3H); 0.26(s,3H);0.96(s.9H); 2.84(m, 2H); 2.87(s,3H); 3.19(m,2H); 3.53(s,3H);3.76(s,3H);d,J=14 Hz, 1H); 4.78(m, 1H); 5.42(s,2H); 6.54(d,J=2 Hz, 1H);6.99(d,J=2 Hz, 1H); 7.2-7.4 (m, 16H); 7.68(d,J=8 Hz,2H); 8.20 d,J=8Hz,2H) ppm

EXAMPLE 34

Crude p-nitrobenzyl2-bromomethyl-3-(tert-butyldimethylsilyloxy)-5-methoxy-6-methylbenzoate,as obtained in Example 23 (d), was subjected in an analogous manner to asequence of procedures described in Example 33 (a, b, d), and theproduct was cyclized and subsequently the silyl groups were cleaved offin in analogous manner to the procedures described in Examples 21 and13, to yield methyl(R)-1,3,4,5,6,7,8,10-octahydro-14-hydroxy-12-methoxy-5,11-dimethyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylateas a white solid.

¹ H-NMR(250 MHz, DMSO-d₆): inter alia; δ1.90 (s, 3H); 2.60 (s, 3H);˜2.80 (m, 2H); 3.00 (dd, J=15 Hz and 5 Hz, 1H); 3.41(d,J=10 Hz, 1H);3.70(s,3H); 3.72 (s,3H); 3.76(d,J=10 Hz, 1H); 6.47(s, 1H); 9.62(s,1H)ppm

EXAMPLE 35

2-[[[(R)-2-[(S)-1-tert-Butoxyformamido)-3-hydroxypropionylamino]-2-(methoxycarbonyl)ethyl]thio]methyl]-3-(tert-butyldimethylsilyloxy)-4-chlorobenzoicacid was cyclized and the tert-butyldimethylsilyl groups subsequentlycleaved off in an analogous manner to the procedure described inExamples 21 and 13. The product obtained was subjected in an analogousmanner to the procedure described in Example 14 (a), the resulting aminewas acylated with N-(tert-butoxycarbonyl)-L-serine in an analogousmanner to the procedure described in Example 14, and the resultingproduct was stirred in trifluoroacetic acid at 0° C. to give, afterevaporation of the solvent and trituration of the residue with diethylether, methyl (4R,7S)-13-chloro-1,3,4,5,6,7,8,10-octahydro-14-hydroxy-6,10-dioxo-7-(L-serylamino)-9,2,5-benzoxathiaazacyclododecine-4-carboxylatetrifluoroacetate as a white solid.

¹ H-NMR(250 MHz, DMSO-d₆): δ2.85(dd, J=14 Hz and 11 Hz, 1H); 3.19(dd,J=14 Hz and 4 Hz, 1H); 3.64(s,3H); ,3.65-3.90(m,2H); 3.98(t,6 Hz, 1H);4.04 (d,J=10 Hz, 1H); 4.34(dd, J=12 Hz and 2 Hz, 1H); 4.42(d,J=10 Hz,1H); 4,50-4.65 (m, 1H); 4.68-4.75 (m, 1H); 4.94 (dd, J=12 Hz and 2 Hz,1H); 6.01(broad t,1H); 7.36(d,J=9 Hz, 1H); 7.44(d,J=9 Hz, 1H); 8.51 (d,J=8 Hz, 1H); 9.11 (d, J=8 Hz, 1H) ppm

The starting material used above was prepared as follows:

(a) To a suspension of 22.8 g of 3-hydroxy-2-methylbenzoic acid in 300ml of water, cooled to 2° C., were added 55 ml of 3N sodium hydroxyde toadjust the pH to 10. To the clear solution were added within 20 minutes,at 2° to 6° C., 125 ml of 1.2N sodium hypochlorite solution, the pHbeing raised to 12.5. Then, 90 ml of 3N hydrochloric acid were added atonce, a precipitate being formed immediately. The mixture was stirred at0° C. for 30 minutes, and subsequently the precipitate was isolated byfiltration and crystallized from diethylether/hexane to give 15.0 g of4-chloro-3-hydroxy-2-methylbenzoic acid as white crystals of m.p. 191°C.

(b) To a solution of 1.86 g 4-chloro-3-hydroxy-2-methylbenzoic acid in20 ml of methylene chloride, cooled to 0° C., were added 3.80 g of g oftert-butyldimethylchlorosilane, 0.12 g of 4-dimethylaminopyridine, and2.25 g of triethylamine. After 5 minutes, the ice-bath was removed, andstirring was continued for 3 hours at 20° C. The mixture was dilutedwith methylene chloride and successively extracted with ice-cold waterand with ice-cold 10% sodium chloride solution. The organic layer wasdried over sodium sulfate, and the solvent was evaporated in vacuo togive 3.5 g of tert-butyldimethylsilyl3-(tert-butyldimethylsilyloxy)-4-chloro-2-methylbenzoate as an oil.

¹ H-NMR (90 MHz,CDCl₃): δ0.24(s,3H); 0.37(s,3H); 1.01(s,9H); 1.04(s,9H);2.52(s,3H); 7.24 (d, J=9 Hz, 1H); 7.55 (d, J=9 Hz, 1H) ppm

(c) tert-Butyldimethylsilyl3-(tert-butyldimethylsilyloxy)-4-chloro-2-methylbenzoate was subjectedin an analogous manner to the procedure described in Example 1(d) togive tertbutyldimethylsilyl2-bromomethyl-3-(tert-butyldimethylsilyloxy)-4-chlorobenzoate as an oil.

¹ H-NMR(90 MHz, CDCl₃): δ0.34(s,3H); 0.42(s,3H); 1.03(s,9H); 1.09(s,9H);4.90 (s, 2H); 7.40(d,J=9 Hz,1H); 7.61 d,J=9 Hz, 1H) ppm

d) To a solution of 5.1 g of tert-butyldimethylsilyl2-bromomethyl-3-(tert-butyldimethylsilyloxy)-4-chlorobenzoate and 3.52 gof N-[N-(tert-butoxycarbonyl)-L-seryl]-L-cysteine methyl ester in 55 mlof methylene chloride was added within 30 minutes, at 0° C., a solutionof 1.59 g of 1,8-diazabicyclo [5.4.0]undec-7-ene (DBU) in 15 ml ofmethylene chloride. Stirring was continued for 3 hours at 0° C., andsubsequently the mixture was diluted with methylene chloride and stirredtogether with 100 ml of 1N aqueous acetic acid. The organic phase waswashed with 10% sodium chloride solution, dried over sodium sulfate, andthe solvent was evaporated in vacuo. The residue was chromatographed onMCI-Gel CHP20P using as eluent a mixture of acetonitrile-watercontaining 40-0% water. The product was eluted with pure acetonitrile.Evaporation of the solvent afforded 2.7 g of2-[[[(R)-2-[(S)-1-tert-butoxyformamido)-3-hydroxypropionylamino]-2-methoxycarbonyl-ethyl ]thio]methyl]-3-(tert-butyldimethylsilyloxy)-4-chlorobenzoic acid as a white foam.

¹ H-NMR(250 MHz, CDCl₃): δ0.27(s,3.H); 0.30(s,3H); 1.05(s,9H);1.46(s,9H); 2.74-3.15 (m, 2H); 3.72(S,3H); 3.81(m, 1H); 4.09(dd, J=12 Hzand 4 Hz,1H); 4.50 (m, 2H); 5.85 (broad d; 1H); 7.18 (d, J=8 Hz, 1H);7.29 (d, J=9 Hz, 1H); 7.50 (d, J=9 Hz, 1H) ppm

EXAMPLE 36

2,4-Dichloro-5-hydroxy-6-methylbenzoic acid was subjected in ananalogous manner to a sequence of reactions described in Example35(b,c,d), and the resulting product was subjected in an analogousmanner to a sequence of reactions used in Example 35 to give, aftercrystallization from diethylether/toluene, methyl (4R,7S)-11,13-dichloro-1,3,4,5,6,7,8,10-octahydro-14-hydroxy-6,10-dioxo-7-(L-serylamino)-9,2,5-benzooxathiaazacyclododecine-4-carboxylateas a white solid.

¹ H-NMR (250 MHz, DMSO-d₆): δ2.86(dd, J=14 Hz and 10 Hz, 1H); 3.21(dd,J=14 Hz and 4 Hz, 1H); 3.60(d,J=10 Hz, 1H); 3.63(s,3H); 3.67-3.84(m,2H); 3.88 (d,J=10 Hz, 1H); 4.03(t,J=5H,1H); 4.15(dd, J=12 Hz and 3 Hz,1H); 4.43-4.56(m, 1H); 4.76-4.84 (m, 1H); 5.31(dd, J=12 Hz and 2 Hz,1H); 5.81(t,J=3H,1H); 7.62(s, 1H); 8.40(d,J=8 Hz,1H); 9.02 (d, J=8 Hz,1H) ppm

The starting material used above was prepared as follows:

(a) To a stirred suspension of 10.0 g of 3-hydroxy-2-methylbenzoic acidin 50 ml of glacial acetic acid, cooled to 2° C., were added 18.8 g ofsulfuryl chloride. Stirring was continued for 5 minutes at 5° C., andfor 30 minutes at 20° C. The solution was concentrated in vacuo to avolume of about 20 ml, and then cooled to 0° C. The precipitate formedwas isolated by filtration, washed with water, and crystallized fromdiethylether/hexane to give 7.2 g of2,4-dichloro-5-hydroxy-6-methylbenzoic acid as white crystals of m.p.135° C.

EXAMPLE 37

3-Hydroxy-2-methylbenzoic acid was subjected in an analogous manner to asequence of reactions described in Example 35(b,c,d), the resultingproduct was cyclized in an analogous manner to the procedure describedin Example 21, the cyclization product was subjected in an analogousmanner to a sequence of reactions described in Example 14(a)/14 to give,after crystallization from methylene chloride/diethylether, methyl (4R,7S)-7-[(S)-2-(1-tert-butoxyformamido)-3-hydroxypropionylamino]-1,3,4,5,6,7,8,10-octahydro-14-hydroxy-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylateas a white solid.

¹ H-NMR(250 MHz, DMSO-d₆): δ1.36(s,9H); 2.70(dd, J=14 Hz and 12 Hz,1H);3.12(dd, J=14 Hz and 4 Hz, 1H); 3.46-3.76(m,2H) superimposed by3.63(s,3H); 3.96 (d, J=10 Hz, 1H); 4.20-4.30 (m, 1H); 4.33 (dd, J=12 Hzand 2 Hz, 1H); 4.45 (d, J=10 Hz, 1H); 4.52-4.72 (m, 2H); 4.85 (dd, J=12Hz and 2 Hz, 1H); 5.52 (t, J=3H, 1H); 6.83 (d, J=8 Hz, 1H); 7.08 (dd,J=8 Hz and 1 Hz, 1H); 7.20 (dd, J=8 Hz and 8 Hz, 1H); 7.36 (dd, J=8 Hzand 1 Hz, 1H); 8.54(d,J=9 Hz,1H); 8.60(d,J=7 Hz, 1H); 10.01(s, 1H) ppm

EXAMPLE 38

Methyl (9S, 12R)-9-amino-6,10-dioxo-2,2-diphenyl-6,8,9,10,11,12,13,15-octahydro-1,3-dioxolo-[4,5-n][9,2,5]-benzooxathiaazacyclododecine-12-carboxylatewas acylated with N-(tert-butoxytcarbonyl)-L-serine in an analogousmanner to the procedure described in Example 14. The product obtainedwas dissolved in 95% aqueous trifluoroacetic acid. The solution wasstirred at room temperature for 75 minutes, and then evaporated invacuo. The residue was was partitioned between ethyl acetate and water.The pH of the concentrated aqueous layer was adjusted to 2.5 by theaddition of 0.5N sodium hydroxide, and the solution was thenchromatographed on MCI-Gel CHP20P using 20% aqueous acetonitrile aseluent. The product-containing fractions were combined and lyophilizedto give methyl (4R,7S)-1,3,4,5,6,7,8,10-octahydro-13,14-dihydroxy-6,10-dioxo-7-(L-serylamino)-9,2,5-benzoxathiaazacyclododecine-4-carboxylateas a white powder.

¹ H-NMR(250MHZ,DMSO-d₆): δ2.75 (dd, 1H); 3.10 (dd, 1H); 3.40-3.70 (m,3H) superimposed by 3.64 (s, 3H); 4.01 (d, 1H); 4.23 (dd, 1H); 4.43 (dd,1H); 4.50-4.70 (m, 2H); 4.75 (dd, 1H); 6.78 (d, 1H); 7.32 (d, 1H); 8.50(d, 1H) ppm

The starting material used above was prepared as follows:

(a) To a suspension of 23.0 g of lithium2,2-diphenyl-1,3-benzodioxole-5-carboxylate in 200 ml ofdimethylformamide were added 27.0 g ofO-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium-hexafluorophosphate.The mixture was stirred at 20° C. for 2 hours, then 25 ml of methylaminewere added, and stirring was continued for another 2 hours. The mixturewas partitioned between ethyl acetate and water, the organic layer waswashed successively with 2N potassium hydrogensulfate solution,saturated sodium carbonate solution and brine, and dried over magnesiumsulfate. The solvent was evaporated in vacuo, and the solid residue wascrystallized from ethanol to give 15.6 g ofN-methyl-2,2-diphenyl-1,3-benzodioxole-5-carboxamide, m.p. 144°-145° C.

(b) To a solution of 3.30 g ofN-methyl-2,2-diphenyl-1,3-benzodioxole-5-carboxamide in 20 ml oftetrahydrofuran, cooled to -78° C., were added 12.5 ml of a 1.6Msolution of n-butyllithium in hexane. The solution was stirred for 1hour, the temperature being allowed to warm up to 20° C. Then, 1.37 g ofmethyl iodide were added, and stirring was continued for 1 hour. Afterthe addition of 20 ml saturated ammonium chloride solution, the mixturewas extracted with ethyl acetate. The organic layer was washedsuccessively with 2N potassium hydrogensulfate solution, saturatedpotassium bicarbonate solution and brine, and dried over magnesiumsulfate. The solvent was evaporated in vacuo, and the solid residue wascrystallized from ethanol to give 15.6 g of N,4-dimethyl-2,2-diphenyl-1,3-benzodioxole-5-carboxamide, m.p. 178°-180°C.

(c) A solution of 1.60 g ofN,4-dimethyl-2,2-diphenyl-1,3-benzodioxole-5-carboxamide and 1.00 g ofsodium hydroxide in 10 ml of ethylene glycol was heated to 190° C. for2.5 hours. After cooling, the mixture was partitioned between water andethyl acetate. The aqueous phase was washed with ethyl acetate, adjustedto pH 2 by the addition of 3N hydrochloric acid, and then the productwas extracted with ethyl acetate. The organic layer was washed withbrine and dried over magnesium sulfate. The solvent was evaporated invacuo, and the solid residue was crystallized from ethanol to give 0.53g of 4-methyl-2,2-diphenyl-1,3-benzodioxole-5-carboxylic acid, m.p.263°-265° C.

(d) 4-Methyl-2,2-diphenyl-1,3-benzodioxole-5-carboxylic acid wassubjected in an analogous manner to a sequence of procedures describedin Examples 1(d, f), 21 and 14(a) to yield, after crystallization fromethyl acetate/hexane, methyl (9S,12R)-9-amino-6,10-dioxo-2,2-diphenyl-6,8,9,10,11,12,13,15-octahydro-1,3-dioxolo-[4,5-n][9,2,5]-benzoxathiaazacyclododecine-12-carboxylateas white solid.

EXAMPLE 39

Crude p-nitrobenzyl 3, 5-bis(tert-butyldimethylsilyloxy)-(α-bromo-2,6-xylate, as obtained in Example13 (e), was reacted with N-[(S)-2,3-dihydroxypropionyl]-L-cysteinemethyl ester in an analogous manner to the procedure described inExample 1(f), and the product was subjected in an analogous manner to asequence of procedures described in Example 13(f) and Example 21 toyield methyl (4R, 7S)-12, 14-bis-(tert-butyldimethylsilyloxy)-1,3,4,5,6,7,8,10-octahydro-7-hydroxy-11-methyl-6,10-dioxo-9,2,5-benzooxathiaazacyclododecine-4-carboxylate.This material was treated with ammonium fluoride in methanol in ananalogous manner to the procedure described in Example 13 to yield,after crystallization from acetonitrile, methyl (4R,7S)-7,12,14-trihydroxy1,3,4,5,6,7,8,10-octahydro-11-methyl-6,10-dioxo-9,2,5-benzooxathiaazacyclododecine-4-carboxylateas a white solid.

¹ H-NMR(250 MHz, DMSO-d₆): δ1.87 (s, 3H); 2.89 (dd, 1H); 3.04 (dd, 1H);3.45 (d, 1H); 3.65 (s, 3H); 3.97 (d, 1H); 4.21 (dd, 1H); 4.33 (m, 1H);4.71 (m, 1H); 5.07 (dd (1H); 6.33 (d, 1H); 6.44 (s, 1H); 8.07 (d, 1H);9.44 (s, 1H); 9.47 (s, 1H) ppm

The starting material used above was prepared as follows:

(a) To a solution of 60.0 g ofsodium-[(S)-2,2-dimethyl-1,3-dioxolan-4-yl]carboxylate in 500 ml ofdimethylformamide were added 136.0 g ofO-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium-hexafluorophosphate.The mixture was stirred at 20° C. for 0.5 hours, then 62 g L-cysteinemethyl ester hydrochloride and subsequently 36.7 g of N-methylmorpholinewere added. The mixture was stirred at 20° C. for 3.5 hours. The solventwas evaporated in vacuo and the oily residue was partitioned betweenwater and ethyl acetate. The organic layer was washed successively withsaturated sodium carbonate solution, saturated potassium hydrogensulfatesolution, and brine, and then dried over magnesium sulfate. The solventwas evaporated in vacuo, and the residual oil was heated at reflux in amixture of 50 ml of trifluoroacetic acid and 500 ml of methanol for 2.5hours. The solvent was evaporated in vacuo, and the residue wascrystallized from tert-butyl-methyl-ether to give 33.4 g ofN-[(S)-2,3-dihydroxypropionyl]-L-cysteine methyl ester as white crystalsof m.p. 90°-92° C.

EXAMPLE 40

Operating in an analogous manner as described in Example 39, but usingin the cyclization step 2 molequivalents of both diethylazodicarboxylate and triphenylphoshine, there was obtained, aftercrystallization from methylene chloride, methyl[(4R,7S)-7-[(ethoxycarbonyl)oxy]-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylateas white crystals of m.p. 224°-225° C.

EXAMPLE 41

Operating in an analogous manner to the sequence of procedures describedin Example 39, but using as starting material crude p-nitrobenzyl2-bromomethyl-3-(tert-butyldimethylsilyloxy)-5-methoxy-6-methylbenzoateas obtained in example 23 (d), there was obtained methyl (4R,7S)-14-(tert-butyldimethylsilyloxy)-1,3,4,5,6,7,8,10-octahydro-7-hydroxy-12-methoxy-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylate. This material was treated with ammonium fluoride inmethanol in an analogous manner to the procedure described in Example 13to yield, after crystallization from ethyl acetate/hexane, methyl (4R,7S)-1,3,4, 5,6,7,8,10-octahydro-7,14-dihydroxy-12-methoxy-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylateas a white solid, m.p. 237°-240° C.

EXAMPLE 42

To a solution of 100 mg of the 12,14-bis-(tert-butyldimethylsilylated)-product of Example 39, 30 mg of2,4-dinitrophenol, and 43 mg of triphenylphosphine in 1 ml of methylenechloride were added 34 mg of diethyl azodicarboxylate. The solution wasstirred at 20° C. for 24 hours, then 43 mg of triphenylphosphine and 34mg of diethyl azodicarboxylate were added, and stirring was continuedfor 72 hours. The solvent was evaporated in vacuo, and the residue waschromatographed on silica gel using ethyl acetate/hexane (1:3, v/v) aseluent. The 12,14-bis-(tert-butyldimethylsilylated)-product obtained wastreated with ammonium fluoride in methanol in an analogous manner to theprocedure described in Example 13 to yield 6 mg of methyl (4R,7S)-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-11-methyl-7-(2,4-dinitro-phenoxy)-6,10-dioxo-9,2,5-benzooxathiaazacyclododecine-4-carboxylateas a white solid.

¹ H-NMR(250 MHz, DMSO-d₆): δ1.85 (s, 3H); 2.72 (dd, 1H); 3.12 (dd, 1H);3.60 (s, 3H); 3.64 (d, 1H); 3.88 (d, 1H) ;4.66(dd, 1H); 4.80(m, 1H);5.19(dd, 1H); 5.77(m, 1H); 6.48 (s, 1H); 7.65 (d, 1H); 8.24 (d, 1H);8.62 (dd, 1H); 8.85 (d, 1H); 9.52 (s, 1H); 9.54 (s, 1H) ppm

EXAMPLE 43

To a solution of 50 mg of the12,14-bis-(tert-butyldimethylsilylated)-product of Example 39 in 1 ml ofmethylene chloride were added at 0° C. 21 mg of pivaloyl chloride and199 mg of 4-dimethylaminopyridine, and the mixture was stirred for 1hour at 20° C. The solvent was evaporated in vacuo, and the residue waschromatographed on silica gel using ethyl acetate/hexane (1:1, v/v) aseluent. The 12,14-bis-(tert-butyldimethylsilylated)-product obtained wastreated with ammonium fluoride in methanol in an analogous manner to theprocedure described in Example 13 to yield methyl (4R,7S)-7-(2,2-dimethylpropionyloxy)-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylate as a whitesolid.

¹ H-NMR (250 MHz, DMSO-d₆): δ1.22(s,9H); 1.86(s,3H); 2.92(dd, 1H);3.10(rid, 1H); 3.59(d, 1H); 3.65 (s, 3H); 3.82 (d, 1H); 4.66 (dd, 1H);4.77 (m, 1H); 4.92 (dd, 1H); 5.39 m, 1H); 6.48(s,1H); 7.74(d, 1H);9.51(s, 1H); 9.56(s,1H) ppm

EXAMPLE 44

Methyl(R)-12,14-bis-(tert-butyldimethylsilyloxy)-1,3,4,5,6,7,8,10-octahydro-11-methyl-6,7,10-trioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylatewas treated with ammonium fluoride in methanol in an analogous manner tothe procedure described in Example 13 to yield, after crystallizationfrom methylene chloride, methyl(R)-12,14-dihydroxy-6,7,10-trioxo-1,3,4,5,6,7,8,10-octahydro-9,2,5-benzoxathiaazacyclododecine-4-carboxylateof a as a white solid, partly in form of its 7-hydrate.

¹ H-NMR(250 MHz, DMSO-d₆): (inter alia) δ1.87(s,3H); 2.62 (dd, 1H); 3.04(dd, 1H); 3.66(s,3H); 3.68 (d, 1H); 3.82 (d, 1H); 4.60 (m, 1H); 4.83 (d,1H); 5.25 (d, 1H); 6.49 (d, 1H); 9.33(d, 1H); 9.57(s,1H); 9.61(s, 1H)ppm; in addition, signals of the 7-hydrated form were observed.

The starting material used above was prepared as follows:

(a) Using in an analogues manner the procedure described in Example16(d), but replacing 3-trityloxy-1-propanol by a sample of 385 mg of the12,14-bis-(tert-butyldimethylsilylated)-product of Example 39, therewere obtained, after chromatographic purification on silica gel usingethyl acetate/hexane (1:1, v/v) as eluent, 184 mg of methyl(R)-12,14-bis-(tert-butyldimethylsilyloxy)-1,3,4,5,6,7,8,10-octahydro-11-methyl-6,7,10-trioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylateas a white solid, possibly in the form of its 7-hydrate.

EXAMPLE 45

A solution of 100 mg of methyl(R)-12,14-bis-(tert-butyldimethylsilyloxy)-1,3,4,5,6,7,8,10-octahydro-11-methyl-6,7,10-trioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylateand 11 mg of hydroxylamine hydrochloride in 2 ml dimethylformamide wasstirred at 20° C. for 3 hours. The solvent was evaporated in vacuo, andthe residue was chromatographed on silica gel using ethyl acetate/hexane(1:2, v/v) as eluent. The12,14-bis-(tert-butyldimethylsilylated)-product obtained was treatedwith ammonium fluoride in methanol in an analogous manner to theprocedure described in Example 13 to yield methyl (E orZ)-(R)-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-7-hydroxyimino-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylateas a white solid.

¹ H-NMR (250 MHz, DMSO-d₆): δ1.88(s,3H); 2.68(dd, 1H); 3.10(dd, 1H);3.60(d, 1H); 3.65(s,3H); 3.75 (d, 1H); 4.72 (m, 1H); 4.98 (d, 1H); 5.56(d, 1H); 6.44 (s, 1H); 8.77 (d, 1H); 9.49 (s. 1H); 9.54 (s, 1H); 11.98(s, 1H) ppm

EXAMPLE 46

Operating in an analogues manner as described in the previous example,but replacing hydroxylamine hydrochloride by O-methyl-hydroxylaminehydrochloride, there was obtained methyl (E orZ)-(R)-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-7-methoxyimino-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylateas a white solid.

¹ H-NMR(250 MHz,DMSO-d₆): δ1.88 (s, 3H); 2.67 (dd, 1H); 3.0 9 (dd, 1H);3.60 (d, 1H); 3.66 (s, 3H); 3.74 (d, 1H); 3.98 (s, 3H); 4.70 (m, 2H);5.00 (d, 1H); 5.52 (d, 1H); 6.45 (s, 1H); 8.96(d, 1H); 9.50(s, 1H);9.55(s, 1H) ppm

EXAMPLE 47

Operating in an analogues manner as described in Example 45, butreplacing hydroxylamine hydrochloride by O-(tert-butyl)hydroxylaminehydrochloride, there was obtained methyl (E orZ)-(R)-7-tert-butoxyimino-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylateas a white solid.

¹ H-NMR(250 MHz, DMSO-d₆): δ1.33(s,9H); 1.88(s,3H); 2.73(dd, 1H);3.11(dd, 1H ; 3.57-3.75 (m, 2H) superimposed by 3.67 (s, 3H); 4.74 (m,1H); 5.06 (d, 1H); 5.47 (d, 1H); 6.46 (s, 1H); 8.73 (d, 1H); 9.50 (s,1H); 9.53 (s, 1H) ppm

EXAMPLE 48

A solution of 100 mg of methyl(R)-14-(tert-butyldimethylsilyloxy)-1,3,4,5,6,7,8,10-octahydro-12-methoxy-11-methyl-6,7,10-trioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylateand 120 mg of triphenylphosporanylidene-acetic acid tertbutylester in 1ml of toluene was stirred at 40° C. for 3 hours. The solvent wasevaporated, and the residue was chromatographed on silica gel usingethyl acetate/hexane (1:3, v/v) as eluent. The twobis-(tert-butyldimethylsilylated)products obtained were treatedseparately with ammonium fluoride in methanol in an analogous manner tothe procedure described in Example 13 to yield

(i) methyl(Z)-(R)-14-hydroxy-1,3,4,5,6,7,8,10-octahydro-12-methoxy-11-methyl-7-tert-butoxycarbonylmethylene-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylateas white crystals, m.p. 146°-149° C. (from acetonitrile), and

(ii) methyl(E)-(R)-14-hydroxy-1,3,4,5,6,7,8,10-octahydro-12-methoxy-11-methyl-7-tert-butoxycarbonylmethylene-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylateas a white solid.

¹ H-NMR(250 MHz, DMSO-d₆) (E-isomer): δ1.49 (s, 9H); 1.91 (s, 3H); 2.67(dd, 1H); 3.12 (dd, 1H); 3.59 (d, 1H); 3.66 (s, 3H); 3.72 (s, 3H); 3.79(d, 1H); 4.68 (m, 1H); 5.27 (d, 1H); 5.89 (d, 1H); 6.13(s,1H); 6.50(s,1H); 8.99(d, 1H); 9.73(s, 1H) ppm

The starting material used above was prepared as follows:

(a) Using in an analogues manner the procedure described in Example16(d), but replacing 3-trityloxy-1-propanol by the14-(tert-butyldimethylsilylated)-product of Example 41, there wasobtained methyl(R)-14-(tert-butyldimethylsilyloxy)-1,3,4,5,6,7,8,10-octahydro-12-methoxy-11-methyl-6,7,10-trioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylate as a whitesolid, partly in the form of its 7-hydrate.

EXAMPLE 49

The product of Example 48 (a) was subjected in an analogues manner tothe procedure described in Example 45, but replacing hydroxylaminehydrochloride by O-methyl-hydroxylamine hydrochloride to yield methyl (Eor Z)(R)-1,3,4,5,6,7,8,10-octahydro-14-hydroxy-12-methoxy-7-methoxyimino-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylateas white crystals of m.p. 234°-236° C. (from acetonitrile).

EXAMPLES 50-57

Using as starting material the products of examples 16, 21, 28, 120,119, 130, 150, and 118, respectively, and operating in an analogousmanner as described in Example 3, the following compounds were prepared:

    __________________________________________________________________________     ##STR19##                                                                    Example                   .sup.1 H-NMR (250 MHz, DMSO-d.sub.6)                No   R.sup.1                                                                          R.sup.2                                                                          R.sup.7        δ, inter alia, ppm                            __________________________________________________________________________    50   Me OH H              1.86(s, 3H); 2.42(m, 1H); 2.58(dd, 1H); 2.91                                  (m, 1H); 3.06(dd, 1H); 3.64(d, 1H); 3.73                                      (d, 1H); 4.44-4.72(m, 3H); 6.42(s, 1H); 8.44                                  (d, 1H); 9.48(d, 1H); 12.86(broad s, 1H)            51   Me OMe                                                                              NHCOOBu.sup.t  1.41(s, 9H); 1.94(s, 3H); 2.91(dd, 1H); 3.05                                  (dd, 1H); 3.72(s, 3H); 3.92(d, 1H); 4.18                                      (dd, 1H); 4.30(m, 1H); 4.44(m, 1H); 4.92                                      (dd, 1H); 6.51(s, 1H); 7.38(d, 2H); 7.89                                      (d, 2H); 9.72(s, 1H)                                52   H  OMe                                                                              NHCOOBu.sup.t  1.42(s, 9H); 2.90(dd, 1H); 3.07(dd, 1H); 3.7                                  1(s, 3H); 3.83(d, 1H); 4.24(dd, 1H); 4.30-                                    4.44(m, 2H); 4.56(m, 1H); 4.80(dd, 1H); 6.59                                  (d, 1H); 6.78(d, 1H); 7.50(d, 1H); 8.08                                       (d, 1H); 10.02(s, 1H); 12.40(broad s, 1H)           53   Me OH NHCOMe         1.90(s, 3H); 1.95(s, 3H); 2.83(dd, 1H); 3.08                                  (dd, 1H); 3.90(d, 1H); 4.10(dd, 1H); 4.36                                     (m, 1H); 4.59(s, 1H); 5.12(dd, 1H); 6.44                                      (s, 1H); 7.94(d, 1H); 8.37(d, 1H); 9.48                                       (s, 1H); 9.50(s, 1H); 12.80(broad s, 1H)            54   Me OMe                                                                              NHCOMe         1.94(s, 3H); 1.95(s, 3H); 2.90(dd, 1H); 3.08                                  (dd, 1H); 3.42(d, 1H); 3.72(s, 3H); 3.86                                      (d, 1H); 4.07(dd, 1H); 4.35(m, 1H); 4.61                                      (m, 1H); 5.11(dd, 1H); 6.51(s, 1H); 8.00                                      (d, 1H); 8.34(d, 1H)9.73(s, 1H); 12.90                                        (s, 1H);                                            55   Me OMe                                                                              NHCHO          1.93(s, 3H); 2.88(dd, 1H):3.07(dd, 1H);                                       3.46(d, 1H); 3.72(s, 3H); 3.84(d, 1H); 4.10                                   (dd, 1H); 4.37(m, 1H); 4.69(m, 1H); 5.17                                      (dd, 1H); 6.51(s, 1H); 8.08(d, 1H); 8.13                                      (s, 1H); 8.62(d, 1H); 9.73(s, 1H)                   56   Me OH NHCH(Me).sub.2 1.01(d, 3H); 1.03(d, 3H); 1.89(s, 3H); 2.81                                   (m, 1H); 3.00(m, 1H); 3.09(d, 1H); 3.87                                       (d, 1H); 4.00-4.18(m, 2H); 4.98(dd, 1H);                                      6.41(s, 1H); 8.36(d, 1H); 9.44(s, 1H); 9.48                                   (s, 1H);                                            57   Me OH                                                                                ##STR20##     1.91(s, 3H); 2.42(s, 3H); 2.52-3.20(m, 7H);                                   3.82(m, 1H); 3.98(dd, 1H); 4.40(m, 1H); 5.20                                  (dd, 1H), 6.46(s, 1H); 7.68(d, 1H); 9.14 (d,                                  1H); 9.52(broad s, 1H)                              __________________________________________________________________________

EXAMPLE 58

To a solution of 114 mg of sodium borohydride in 1.5 ml of 80% aqueousmethanol, cooled to 0° C., was added within 5 minutes a solution of 214mg of tert-butyl (4R,7S)-12,14-bis(tert-butyldimethylsilyloxy)-1,3,4,5,6,7,8,10-octahydro-4-(methoxycarbonyl)-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamatein 1.5 ml of methanol/tetrahydro-furan (1:1, v/v). The solution wasstirred at 0° C. for 30 minutes, and then poured into ice-cold 0.5Nhydrochloric acid. The mixture was extracted with ethyl acetate, and theorganic layer was washed with brine, and dried over sodium sulfate. Thesolvent was evaporated in vacuo to yield 181 mg of tert-butyl (4R,7S)-12, 14-bis (tert-butyldimethylsilyloxy)-1,3,4,5,6,7,8,10-octahydro-4-hydroxymethyl-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamateas an amorphous solid. This material was treated with ammonium fluoridein methanol in an analogous manner to the procedure described in Example13 to yield, after crystallization from ethyl acetate/methanol/hexane,tert-butyl (4R, 7S)-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-4-hydroxymethyl-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamateas a white solid.

¹ H-NMR(250 MHz, DMSO-d₆): δ1.41(s,9H); 1.89,(s,3H); 2.53(dd, 1H);2.83(dd, 1H); 3.24-3.50 (m, 3H); 3.74-3.93(m, 2H); 4.16(dd, 1H); 4.30(m,1H); 4.79(t,1H); 4.93(dd, 1H); 6.44(s, 1H); 7.13(d, 1H); 7.60(d, 1H);9.45(s, 1H); 9.49 (s, 1H) ppm

EXAMPLES 59-61

Using as starting material the 12,14-bis(tert-butyldimethylsilylated)-product of Example 16, and the14-(tert-butyldimethylsilylated)-products of Examples 21 and 32,respectively, and operating in an analogous manner as described inExample 58, the following compounds were prepared.

    __________________________________________________________________________     ##STR21##                                                                    Example                .sup.1 H-NMR (250 MHz, DMSO-d.sub.6)                   No   R.sup.1                                                                          R.sup.2                                                                          R.sup.7                                                                              R.sup.8                                                                            δ, inter alia, ppm                               __________________________________________________________________________    59   Me OH H      H    1.86(s, 3H); 2.33(m, 1H); 2.75-2.90                                           (m, 1H); 3.20-3.45(m, 2H); 3.61(d, 1H)                                        3.70(d, 1H); 3.94(m, 1H); 4.48(m, 1H);                                        4.64(m, 1H); 4.78(t, 1H); 6.41(s, 1H);                                        7.86(d, 1H); 9.40(s, 1H); 9.46(s, 1H)                  60   Me OMe                                                                              NHCOOBu.sup.t                                                                        H    1.41(s, 9H); 1.93(s, 3H); 2.83(dd, 1H);                                       3.26-3.42(m, 2H); 3.45(d, 1H); 3.72                                           (s, 3H); 3.85(d, 1H); 4.15(dd, 1H); 4.27                                      (m, 1H); 4.79(t, 1H); 4.95(dd, 1H); 6.51                                      (s, 1H); 7.13(d, 1H); 7.59(d, 1H); 9.68                                       (s, 1H)                                                61   H  OMe                                                                              H      CH.sub.2 OH                                                                        2.30-2.72(m, 4H); 3.22-3.42(m, 2H);                                           3.52(t, 1H); 3.70(s, 3H); 3.83(d, 1H);                                        4.02(m, 1H); 4.52(d, 1H); 4.78(t, 1H);                                        4.98(t, 1H); 5.46(m, 1H); 6.55(d, 1H);                                        6.61(d, 1H); 7.98(d, 1H); 9.91(s,                      __________________________________________________________________________                           1H)                                                

EXAMPLE 62

A solution of 50 mg of tert-butyl (4R,7S)-12,14-bis(tertbutyldimethylsiiyloxy)-1,3,4,5,6,7,8,10-octahydro-4-hydroxy-methyl-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclodecine-7-carbamatein 1 ml of acetic anhydride was treated with 20 mg of pyridine, and thenstirred for 3 hours at 20° C. The solution was evaporated in vacuo, andthe residue was dissolved in ethyl acetate. The solution was washed with5% sodium bicarbonate solution and with brine, and then dried oversodium sulfate. The solvent was evaporated in vacuo to yield 52 mg oftert-butyl(4R,7S)-4-acetoxymethyl-12,14-bis(tert-butyldimethylsilyloxy)-1,3,4,5,6,7,8,10-octahydro-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7carbamate.This material was treated with ammonium fluoride in methanol in ananalogous manner to the procedure described in Example 13 to yield,after crystallization from ethyl acetate/methanol/hexane, tert-butyl(4R, 7S)-4-acetoxymethyl-1,3,4,5,6,7,8,10 -octahydro-12,14-dihydroxy-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamateas a white solid.

¹ H-NMR (250 MHz, DMSO-d₆): δ1.41 (s, 9H); 1.89 (s, 3H); 1.98 (s, 3H);2.53 (dd, 1H); 2.82 (dd, 1H); 3.47 (d, 1H); 3.82 (d, 1H); 3.90-4.16 (m,3H); 4.21 (dd, 1H); 4.32 (m, 1H); 4.95 (m, 1H); 6.44 (s, 1H); 7.02 (d,1H); 7.78 (d, 1H); 9.48 (s, 1H); 9.51 (s, 1H) ppm

EXAMPLE 63

The 12,14-bis (tert-butyldimethylsilylated)-product of Example 59 wassubjected in an analogous manner to the procedure described in Example62 to give[(R)-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecin-4-yl]-methylacetate as a white solid.

¹ H-NMR (250 MHz, DMSO-d₆): δ1.88(s,3H); 2.01(s,3H); 2.33-2.47(m,2H);2.75-2.89(m, 2H); 3.69 (dd, 2H); 3.89 (dd, 1H); 3.99 (dd, 1H); 4.17 (m,1H); 4.51 (m, 1H); 4.61 (m, 1H); 6.41(s, 1H); 8.05(d, 1H); 9.43(s,1H);9.48(s, 1H) ppm

EXAMPLE 64

The 14-(tert-butyldimethylsilylated)-product of Example 60 was subjectedin an analogous manner to the procedure described in Example 62 to givetert-butyl (4R,7S)-4-acetoxymethyl-1,3,4,5,6,7,8,10-octahydro-14-hydroxy-12-methoxy-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamateas a white solid.

¹ H-NMR(250 MHz, DMSO-d₆): δ1.41 (s, 9H); 1.92 (s, 3H); 1.98 (s, 3H);2.53 (dd, 1H); 2.83 (dd, 1H); 3.50 (d, 1H); 3.72 (s,3H); 3.86(d, 1H);3.90-4.15(m, 1H); 4.20(dd, 1H); 4.29 (m, 1H); 4.95(m, 1H); 6.51(s, 1H);7.01(d, 1H); 7.79(d, 1H); 9.72 (s, 1H) ppm

EXAMPLE 65

A solution of 74 mg of the12,14-bis(tert-butyldimethylsilylated)-product of Example 59, 23 mg ofphtalic anhydride, and 19 mg of 4-dimethylaminopyridine in 2 ml ofmethylene chloride was heated at reflux for 3 hours. The solution wasdiluted with methylene chloride, washed with 3N hydrochloric acid andwith brine, and then dried over sodium sulfate. The solvent wasevaporated in vacuo, the residue was taken up in 2.6 ml of methanol, and26 mg of ammonium fluoride were added. The mixture was stirred for 30minutes at 20° C., and then partitioned between water and ethyl acetate.The aqueous phase was concentrated and chromatographed on MCI-Gel CHP20Pusing 0-20% aqueous acetonitrile as eluent. The product-containingfractions were combined and lyophilized to give[(R)-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-11-methyl-6,10-dioxo9,2,5-benzoxathiaazacyclododecin-4-yl]-methylhydrogen phtalate as a white powder.

¹ H-NMR(250 MHz, DMSO-d₆): δ1.67(s,3H); 2.22-2.56(m, 2H); 2.77-2.97(m,2H); 3.70(m, 2H); 4.10-4.40 (m, 3H); 4.47-4.71 (m, 2H); 6.42 (s, 1H);7.60-7.80 (m, 4H); 8.17 (d, 1H); 9.43(s,1H); 9.48(s, 1H); 13.35(broad s,1H) ppm

EXAMPLE 66

A solution of 103 mg of tert-butyl (4R, 7S)-12, 14-bis(tertbutyldimethylsilyloxy)-1,3,4,5,6,7,8,10-octahydro-4-hydroxymethyl-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamatein 2 ml of methylene chloride was treated with 99 mg ofcarbontetrabromide and 79 mg of triphenylphosphine, and then stirred at20° C. for 15 minutes. The solution was chromatographed on silica gelusing ethyl acetate/hexane (1:3, v/v) as eluent to yield tert-butyl (4R,7S)-4-bromomethyl-12,14-bis-(tert-butyldimethylsilyloxy)-1,3,4,5,6,7,8,10-octahydro-11-methyl-6,10-dioxo-9,2,5-benzooxathiaazacyclododecine-7-carbamate.This material was treated with ammonium fluoride in methanol in ananalogous manner to the procedure described in Example 13 to yield,after crystallization from ethyl acetate/hexane, tert-butyl (4R,7S)-4-bromomethyl-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamateas a white solid.

Mass spectrum: m/z (inter alia) 540 (M+Na); 465/463 (M+H-C₄ H₈)

EXAMPLE 67

A solution of 61 mg of tert-butyl (4R,7S)-14-(tert-butyldimethylsilyloxy)-1,3,4,5,6,7,8,10-octahydro-12-methoxy-4-methoxycarbonyl-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamateand 11.4 mg of tetraisopropyl orthotitanate in 2 ml of ethanol washeated at reflux for 40 hours. The solution was diluted with ethylacetate and washed successively with 1N hydrochloric acid and withbrine. The organic layer was dried over sodium sulfate and evaporated invacuo. The residue was chromatographed on silica gel using ethylacetate/hexane (1:2, v/v) as eluent to yield tert-butyl (4R,7S)-14-(tert-butyldimethylsilyloxy)-4-ethoxycarbonyl-1,3,4,5,6,7,8,10-octahydro-12-methoxy-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamate.This material was treated with ammonium fluoride in methanol in ananalogous manner to the procedure described in Example 13 to yield,after crystallization from ethyl acetate/hexane, tert-butyl (4R,7S)-4-ethoxycarbonyl-1,3,4,5,6,7,8,10-octahydro-14-hydroxy-12-methoxy-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamateas a white solid.

¹ H-NMR(250 MHz,DMSO-d₆): δ1.17(t,J=7 Hz,3H); 1.41(s,9H); 1.93(s,3H);2.85(dd, J=14 Hz and 10 Hz, 1H); 3.03 (dd, J=14 Hz and 4 Hz, 1H); 3.43(d, J=10 Hz, 1H); 3.72 (s,3H); 3.83(d,J=10 Hz, 1H); 4.09(q,J=7 Hz,2H);4.21(dd, J=10 Hz and 4 Hz, 1H); 4.34 (m, 1H); 4.48 (m, 1H); 4.85 (m,1H); 5.51 (s, 1H); 7,26(d,J=8 Hz, 1H); 8.14(d,J=SHz, 1H); 9.73(s, 1H)ppm

EXAMPLES 68-77

Operating in an analogues manner as described in the previous example,the product of Example 2 was transesterfied with n-propanol (1 hour at100° C.), the product of Example 130 with ethanol (24 hours at reflux),the 14-(tert,butyldimethylsilylated)-product of Example 21 with2-methoxy-ethanol (20 hours 120° C.),with cyclopropylmethanol (40 hoursat 120° C.), and with 4-hydroxy-tetrahydropyran (36 hours at 100° C.),the 14-(tert-butyldimethylsilylated)-product of Example 130 with2-methoxy-ethanol (20 hours 120° C.), and the12,14-bis-(tert-butyldimethylsilylated)-product of Example 13 withcyclopentanol (36 hours at 120° C.), with 3-butin-1-ol (48 hours atreflux), and with 2,5-dimethoxybenzyl alcohol (48 hours at 120° C.), andthe 12,14-(tert-butyldimethylsilylated)-product of Example 16 with allylalcohol to give the following compounds:

    __________________________________________________________________________     ##STR22##                                                                    Example                      .sup.1 H-NMR (250 MHz, DMSO-d.sub.6)             No   R.sup.2                                                                          R.sup.5       R.sup.7                                                                              δ, inter alia, ppm                         __________________________________________________________________________    68   OH COOCH.sub.2 CH.sub.2 CH.sub.3                                                               NHCOOBu.sup.t                                                                        0.87(t, 3H); 1.41(s, 9H); 1.57(q, 2H);                                        1.90(s, 3H); 2.87(dd, 1H); 3.04(dd, 1H);                                      3.42(d, 1H); 3.79(d, 1H); 4.00(t, 2H);                                        4.19(dd, 1H); 4.34(m, 1H); 4.47(m, 1H);                                       4.83(m, 1H); 6.41(s, 1H); 7.21(d, 1H);                                        8.13(d, 1H); 9.49(s, 1H); 9.51(s, 1H)            69   OMe                                                                              COOCH.sub.2 CH.sub.3                                                                        NHCHO  1.17(t, 3H); 1.93(s, 3H); 2.87(dd, 1H);                                       3.09(dd, 1H); 3.57(d, 1H); 3.72(s, 3H);                                       3.76(d, 1H); 4.07(q, 2H); 4.13(dd, 1H);                                       4.39(m, 1H); 4.71(m, 1H); 5.10(dd, 1H);                                       6.49(s, 1H); 8.10(s, 1H); 0.31(d, 1H);                                        8.53(d, 1H); 9.68(s, 1H)                         70   OMe                                                                              COOCH.sub.2 CH.sub.2 OCH.sub.3                                                              NHCOOBu.sup.t                                                                        1.41(s, 9H); 1.93(s, 3H); 2.81(dd, 1H)                                        3.05(dd, 1H); 3.25(s, 3H); 3.42-3.54                                          (m, 3H); 3.72(s, 3H); 3.82(d, 1H); 4.07-                                      4.25(m, 3H); 4.35(m, 1H); 4.48(m, 1H);                                        4.87(dd, 1H); 6.51(s, 1H); 7.20(d, 1H)                                        8.19(d, 1H); 9.73(s, 1H)                         71   OMe                                                                               ##STR23##    NHCOOBu.sup.t                                                                        0.26(m, 2H); 0.50(m, 2H); 1.07(m, 1H)                                         1.41(s, 9H); 1.93(s, 3H); 2.87(dd, 1H);                                       3.06(dd, 1H); 3.45(d, 1H); 3.72(s, 3H);                                       3.84(d, 1H); 3.89(m, 2H); 4.20(dd, 1H);                                       4.35(m, 1H); 4.49(m, 1H); 4.85(m, 1H);                                        6.51(s, 1H); 7.24(d, 1H); 8.14(d, 1H)                                         9.74(s, 1H)                                      72   OMe                                                                               ##STR24##    NHCOOBu.sup.t                                                                        1.41(s, 9H); 1.48-1.64(m, 2H); 1.75- 1.91(m,                                  2H); 1.93(s, 3H); 2.89(dd, 1H) 3.06(dd, 1H);                                  3.38-3.54(m, 3H); 3.66- 3.87(m, 3H)                                           superimposed by 3.72 (s, 3H); 4.25(dd, 1H);                                   4.32-4.52(m, 2H); 4.81(dd, 1H); 4.89(m, 1H);                                  6.50(s, 1H) 7.26(d, 1H); 8.25(d, 1H);                                         9.73(s, 1H)                                      73   OMe                                                                              COOCH.sub.2 CH.sub.2 OCH.sub.3                                                              NHCHO  1.93(s, 3H); 2.87(dd, 1H); 3.10(dd, 1H);                                      3.25(s, 3H); 3.51(t, 2H); 3.57(d, 1H)                                         3.72(s, 3H); 3.74(d, 1H); 4.07-4.23                                           (m, 3H); 4.40(m, 1H); 4.72(m, 1H); 5.11                                       (dd, 1H); 6.51(s, 1H); 8.12(s, 1H); 8.39                                      (d, 1H); 8.53(d, 1H); 9.70(broad s, 1H)          74   OH                                                                                ##STR25##    NHCOOBu.sup.t                                                                        1.41(s, 9H); 1.44-1.86(m, 9H); 1.89 (s, 3H);                                  2.86(dd, 1H); 3.02(dd, 1H); 3.34 (s, 3H);                                     3.41(d, 1H); 3.76(d, 1H); 4.21 (dd, 1H);                                      4.30-4.45(m, 2H); 4.79m, 1H); 5.06(m, 1H);                                    6.41(s, 1H); 7.27(d, 1H); 8.15(d, 1H);                                        9.48(s, 1H); 9.50(s, 1H)                         75   OH COOCH.sub.2 CH.sub.2 CCH                                                                    NHCOOBu.sup.t                                                                        1.41(s, 9H); 1.89)s, 3H); 2.83(dd, 1H);                                       2.90(t, 1H); 3.04(dd, 1H); 3.48(d, 1H);                                       3.77(d, 1H); 4.11(m, 2H); 4.20(dd, 1H);                                       4.36(m, 1H); 4.48(m, 1H); 4.85(dd, 1H);                                       6.43(s, 1H); 7.18(d, 1H); 8.26(d, 1H);                                        9.49(s, 1H); 9.51                                76   OH                                                                                ##STR26##    NHCOOBu.sup.t                                                                        1.39(s, 9H); 1.89(s, 3H); 2.87(dd, 1H);                                       3.06(dd, 1H); 3.41(d, 1H); 3.70(s, 3H);                                       3.74(s, 3H); 3.81(d, 1H); 4.15(dd, 1H);                                       4.34(m, 1H); 4.56(m, 1H); 4.88(dd, 1H);                                       5.03(d, 1H); 5.13(d, 1H); 6.45(s, 1H)                                         6.84-6.98(m, 3H); 7.20(d, 1H); 8.21 (d, 1H);                                  9.50(s, 1H); 9.52(s, 1H)                         77   OH COOCH.sub.2 CHCH.sub.2                                                                      H      1.86(s, 3H); 2.60(dd, 1H); 2.90(m, 1H)                                        3.08(dd, 1H); 3.65-3.54(m, 3H); 4.45-                                         4.75(m, 6H); 5.30(dd, 1H); 5.38(dd, 1H)                                       5.90(m, 1H); 6.42(s, 1H); 8.60(d, 1H)                                         9.46(s, 1H); 9.50(s, 1H)                         __________________________________________________________________________

EXAMPLE 78

A solution of 291 mg of the product of Example 2 was dissolved in 6 mlof saturated methanolic ammonia which had been prepared by theintroduction of dry ammonia into methanol at 10° C. The solution waskept at 20° C. for 7 hours, and then evaporated in vacuo. The residuewas crystallized from ethyl acetate to yield 243 mg of tert-butyl (4R,7S)-4-carbamoyl-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamateas a white solid.

¹ H-NMR(250 MHz, DMSO-d₆): δ1.41 (s, 9H); 1.90 (s, 3H); 2.75 (dd, 1H);3.01 (dd, 1H); 3.32 (d, 1H); 3.89 (d, 1H); 4.15(dd, 1H); 4.22(m, 1H);4.39(m, 1H); 4.93(dd, 1H); 6.44 (s, 1H); 7.25 (s, 1H); 7.38 (s, 1H);7.54 (d, 1H); 7.88 (d, 1H); 9.46 (s, 1H); 9.49 (s, 1H) ppm

EXAMPLE 79

The product of Example 32 was subjected in an analogous manner to theprocedure described in the previous example to yield, aftercrystallization from ethyl acetate/hexane,(4R,8S)-1,3,4,5,6,7,8,10-octahydro-14-hydroxy-12-methoxy-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4,8-dicarbamateas a white solid.

¹ H-NMR(250 MHz,DMSO-d₆): δ2.85 (dd, 1H); 3.03 (dd, 1H); 3.72 (s. 3H);3.90 (d, 1H); 4.57-4.71 (m, 2H); 5.52(dd, 1H); 6.61(d, 1H); 6.76(d, 1H);6.93(s,1H); 7.24 (s, 1H); 7.50(s, 1H); 7.73(s,1H); 8.56(d, 1H);10.03(s,1H) ppm

EXAMPLE 80

A solution of 40 mg of the product of Example 39 in 0.5 ml of methanoland 0.1 ml of 40% aqueous methylamine was stirred at 20° C. for 1 hour.The solution was evaporated in vacuo, and the residue waschromatographed on silica gel using ethyl acetate/methanol (9:1, v/v) aseluent. The purified product was triturated with chloroform to yield 20mg of (4R,7S)-7,12,14-trihydroxy-1,3,4,5,6,7,8,10-octahydro-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylic acid methylamide as a white solid, m.p. 275°-277° C.

¹ H-NMR (250 MHz,DMSO-d₆): δ1.87 (s, 3H); 2.59 (d, 3H); 2.73 (dd, 1H);2.99 (dd, 1H); 3.32 (s, 3H); 3.38 (d, 1H); 3.93(d, 1H); 4.18(dd, 1H);4.29(m, 1H); 4.48(m, 1H); 5.07 (dd, 1H); 6.25 (d, 1H); 6.43 (s, 1H);7.79 (d, 1H); 7.91 (d, 1H); 9.44 (d, 1H) ppm

EXAMPLE 81

A solution of 154 mg of the product of Example 119 and 1.52 g ofallylamine in 2 ml of methanol was heated to 50° C. for 2 hours. Thesolution was evaporated in vacuo, and the residue was dissolved in 3 mlof methanol. The solution was diluted with ethyl acetate and washedsuccessively with 3N hydrochloric acid and with brine. The organic layerwas dried over sodium sulfate and evaporated in vacuo. The residue wascrystallized from methanol/diethylether to give 85 mg of (4R,7S)-7-acetylamino-1,3,4,5,6,7,8,10-octahydro-14-hydroxy-12-methoxy-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylicacid allylamide as a white solid.

¹ H-NMR (250 MHz, DMSO-d₆): δ1.93(s,3H); 1.96(s,3H); 2.78(dd, 1H);3.08(dd, 1H); 3.40(d, 1H); 3.65-3.88(m, 2H) superimposed by 3.72(s,3H);3.93(d, 1H); 4.11 (dd, 1H); 4.38-4.51 (m, 2H); 5.05 (dd, 1H); 5.13 (dd,1H); 5.19 (dd, 1H); 5.78 (m, 1H); 6.52 (s, 1H); 7.85 (d, 1H); 7.96 (t,1H); 8.51 (d, 1H); 9.73 (s, 1H) ppm

EXAMPLE 82

To a solution of 188 mg of the product of Example 3 in 6 ml ofacetonitrile, cooled to 0° C., were added 46 mg of N-hydroxysuccinimideand 83 mg of dicyclohexyl-carbodiimide. The mixture was stirred at 0° C.for 3 hours, and then allowed to warm up to 20° C. within 1 hour. Asolution of 144 mg of L-alanine 2,2,2-trichloroethylester in 1 ml ofacetonitrile was added to the mixture, and stirring was continued at 20°C. for 1.5 hours. The precipitate was removed by filtration, and thefiltrate was diluted with ethyl acetate and washed successively with 3Nhydrochloric acid, water, saturated sodium carbonate solution, andbrine. The organic layer was dried over sodium sulfate and evaporated invacuo. The residue was chromatographed on silica gel using ethylacetate/hexane (2:1, v/v) as eluent to yield 156 mg of N-[[(4R,7S)-1,3,4,5,6,7,8,10-octahydro-12,14-hydroxy-7-(tert-butoxycarbonylamino)-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecin-4-yl]-carbonyl]-L-alanine2,2,2-trichloroethylester. A sample of 121 mg of this material wassubjected in an analogous manner to the procedure described in Example 1(g) to yield after crystallization from ethyl acetate/hexane 90 mg ofN-[[(4R,7S)-1,3,4,5,6,7,8,10-octahydro-12,14-hydroxy-7-(tertbutoxycarbonylamino)-11-methyl-6,10-dioxo-9,2,5-benzooxathiaazacyclododecin-4-yl]-carbonyl]-L-alanineas a white solid.

¹ H-NMR(250 MHz, DMSO-d₆): δ1.24 (d, 1H); 1.41 (s, 9H); 1.90 (s, 3H);2.64 (dd, 1H); 3.01 (dd, 1H); 3.38 (d, 1H); 3.92 (d, 1H); 4.00-4.18 (m,2H); 4.30 (m, 1H); 4.58m, 1H); 4.97 (dd, 1H); 6.45 (s, 1H); 7.43(d, 1H);7.94(d, 1H); 8.23 (d, 1H); 9.52(s,1H); 9.55(s, 1H) ppm

EXAMPLE 83

To a suspension of 107 mg of the product of Example 54 in 5 ml ofacetonitrile, cooled to 0° C., were added 43 mg of N-hydroxysuccinimideand 72 mg of N-(dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride.The mixture was stirred at 0° C. for 3 hours, then 68 mg of β-alaninetert-butylester hydrochloride and 38 mg of 4-methylmorpholine wereadded, and stirring was continued for for 15 hours at 20° C. The mixturewas diluted with ethyl acetate and washed successively with 3Nhydrochloric acid, water, saturated sodium bicarbonate solution, andbrine. The organic layer was dried over sodium sulfate and evaporated invacuo. The residue was dissolved in 2.5 ml of trifluoroacetic acid, andthe solution was stirred at 0° C. for 30 minutes. The solvent wasevaporated in vacuo, and the residue was taken up in 1 ml of water andchromatographed on MCI-Gel CHP20P using 0-20% aqueous acetonitrile aseluent. The product-containing fractions were combined and lyophilizedto give 9 mg ofN-[[(4R,7S)-7-acetylamino-1,3,4,5,6,7,8,10-octahydro-14-hydroxy-12-methoxy-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-yl]-carbonyl.beta.-D-alanineas a white powder.

¹ H-NMR (250 MHz, DMSO-d₆): δ1.82-2.09 (m, ˜3H) superimposed by 1.93 (s,3H) and 1.98 (s. 3H); 2.66 (dd, 1H); 3.04-3.22 (m, 3H); 3.59 (d, 1H);3.71 (s, 3H); 3.73 (d, 1H); 4.45 (m, 1H); 4.75 (dd, 1H); 6.52 (s, 1H);8.39 (d, 1H); 8.68 (t, 1H); 9.15 (d, 1H); 10.10 (broad s, 1H) ppm

EXAMPLE 84

To a solution of 97 mg of the product of Example 51 in 6 ml ofacetonitrile, cooled to 0° C., were added 34.5 mg ofN-hydroxysuccinimide and 57.5 mg ofN-(dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride. The mixturewas stirred at 0° C. for 3 hours, then a solution of 23 mg of allylaminein 1 ml of acetonitrile was added, and stirring was continued for 1.5hours at 20° C. The mixture was diluted with ethyl acetate and washedsuccessively with 3N hydrochloric acid, water, saturated sodiumcarbonate solution, and brine. The organic layer was dried over sodiumsulfate and evaporated in vacuo. The residue was chromatographed onsilica gel using ethyl acetate/hexane (2:1, v/v) as eluent to yield,after crystallization from ethyl acetate/hexane, 30 mg of(4R,7S)-7-tert-butoxycarbonylamino-1,3,4,5,6,7,8,10-octahydro-14-hydroxy-12-methoxy-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylicacid allylamide as a white solid.

¹ H-NMR (250 MHz, DMSO-d₆): δ1.82-2.09 (m, 2H) superimposed by 1.93 (s,3H) and 1.98 (s, 3H); 2.66 (dd, J=13 Hz and 10 Hz, 1H); 3.04-3.22 (m,3H); 3.59 (d,J=10 Hz, 1H); 3.71(s,3H); 3.73(d,J=10 Hz, 1H); 4.20-4.37(m, 2H); 4.45(m, 1H); 4.75(m, 1H); 6.52(s, 1H); 8.39(d,J=8 Hz, 1H); 8.68(t, J=5 Hz, 1H); 9.15 (d, J=7 Hz, 1H); 10.10 (broad s, 1H) ppm

EXAMPLES 85-91

Operating in an analogues manner as described in the previous example,but reacting the product of Example 3 with decylamine, the product ofExample 54 with β-alanine methylester, and the product of Example 51with aminomethyl-cyclohexane, propargyl-amine,(4-trifluoromethyl-benzyl)-amine, ethyl4-amino-1-piperidine-carboxylate, and cyclopentylamine, respectively,the following compounds were obtained.

    __________________________________________________________________________     ##STR27##                                                                    Example                         .sup.1 H-NMR (250 MHz, DMSO-d.sub.6)          No   R.sup.2                                                                          R.sup.5          R.sup.7                                                                              δ, inter alia, ppm                      __________________________________________________________________________    85   OH CONH(CH.sub.2).sub.9 CH.sub.3                                                                  NHCOOBu.sup.t                                                                        1.1-1.5(m, 16H) superimposed by                                               1.41(s, 9H); 1.89(s, 3H); 2.68(dd, 1H);                                       2.91-3.13(m, 3H); 3.33(s, 3H); 3.38(d,                                        1H); 3.89(d, 1H); 4.16(dd, 1H); 4.25(m,                                       1H); 4.35(m, 1H); 4.96(dd, 1H)6.44(s,                                         1H); 7.50(d, 1H); 7.84(t, 1H); 7.92(d,                                        1H); 9.48(s, 1H); 9.50(s, 1H)                 86   OMe                                                                               ##STR28##       NHCOMe 1.93(s, 3H); 1.96(s, 3H); 2.70(dd, 1H);                                       3.02(dd, 1H); 3.20-3.44(m, 2H); 3.60 (s,                                      3H); 3.72(s, 3H); 3.92(d, 1H); 4.06 (dd,                                      1H); 4.30-4.58(m, 2H); 5.18 (dd, 1H);                                         6.49(s, 1H); 7.77(d, 1H); 7.88(t, 1H);                                        8.49(d, 1H); 9.71(s, 1H)                      87   OMe                                                                               ##STR29##       NHCOOBu.sup.t                                                                        0.85(m, 2H); 1.39(m, 3H); 1.3-1.5 (m, 1H)                                     superimposed by 1.41(s, 9H) 1.93(s, 3H);                                      2.69(dd, 1H); 2.82-3.10 (m, 3H); 3.38(d,                                      1H); 3.72(s, 3H); 3.92 (d, 1H); 4.17(dd,                                      1H); 4.25(m, 1H); 4.47 (m, 1H); 4.96(dd,                                      1H); 6, 51(s, 1H); 7.49 d, 1H);                                               7.77-7.97(m, 2H); 9.70(s, 1H)                 88   OMe                                                                               ##STR30##       NHCOOBu.sup.t                                                                        1.42(s, 9H); 1.93(s, 3H); 2.66(dd, 1H)                                        3.00(dd, 1H); 3.00(dd, 1H); 3.14(t, 1H) ;                                     3.43(s, 1H); 3.72(s, 3H); 3.86 (m, 2H);                                       3.91(d, 1H); 4.17(dd, 1H); 4.29 (m, 1H);                                      4.49(m, 1H); 4.94(dd, 1H); 6.51 (s, 1H);                                      7.42(d, 1H); 7.98(d, 1H); 8.38 (t, 1H);                                       9.71(s, 1H)                                   89   OMe                                                                               ##STR31##       NHCOOBu.sup.t                                                                        1.36(s, 9H); 1.93(s, 3H); 2.76(dd, 1H);                                       3.08(dd, 1H); 3.41(d, 1H); 3.72(s, 3H);                                       3.85(d, 1H); 4.20(d, 1H); 4.32(m, 1H);                                        4.40(m, 2H); 4.54(m, 1H); 4.98(dd, 1H);                                       6.52(s, 1H); 7.46(m, 3H); 7.67(d, 2H);                                        8.08(d, 1H); 8.52(m, 1H); 9.73(s, 1H)         90   OMe                                                                               ##STR32##       NHCOOBu.sup.t                                                                        1.17(t, 3H); 1.41(s, 9H); 1.60-1.78 (m,                                       2H); 2.68(dd, 1H); 2.80-3.04 (m, 3H);                                         3.70-3.94(m, 2H) superim- posed by                                            3.72(s, 3H); 4.01(q, 2H); 4.16(dd, 1H);                                       4.24(m, 1H); 4.46(m, 1H); 4.96(dd, 1H);                                       6.51(s, 1H); 7.51(d, 1H); 7.85(d, 1H);                                        7.90(d, 1H); 9.71(s, 1H)                      91   OMe                                                                               ##STR33##       NHCOOBu.sup.t                                                                        1.25-1.87(17H); 1.93(s, 3H); 2.65 (dd,                                        1H); 2.95(m, 1H); 3.72(s,                                                     3H); 3.85-4.02(m, 2H); 4.14(dd, 1H); 4.22                                     m, 1H); 4.43(m, 1H); 4.98(dd, 1H);                                            6.51(s, 1H); 7.52(d, 1H); 7.76- 7.92(m,                                       2H); 9.71(s, 1H)                              __________________________________________________________________________

EXAMPLE 92

A solution of 97 mg of the product of Example 51, 105 mg oftriphenylphosphine, and 67 mg of dipyridyl-2,2-disulfide in 2 ml ofmethylene chloride were stirred at 20° C. for 30 minutes, then 75 mg of3-amino-1-methoxybenzene were added, and stirring was continued for 1hour. The mixture was diluted with ethyl acetate and washed successivelywith 0.1N sodium hydroxyde solution, 1N hydrochloric acid, and withbrine. The organic layer was dried over sodium sulfate and evaporated invacuo. The residue was chromatographed on silica gel using methylenechloride/ethyl acetate (2:1, v/v) as eluent to yield, aftercrystallization from ethyl acetate/hexane 40 mg of (4R,7S)-7-tert-butoxycarbonylamino-1,3,4,5,6,7,8,10-octahydro-14-hydroxy-12-methoxy-11-methyl-6,10-dioxo-9,2,5-benzooxathiaazacyclododecine-4-carboxylicacid 3-methoxyphenylamide as a white solid.

¹ H-NMR (250 MHz, DMSO-d₆): δ1.42(s,9H); 1.94(s,3H); 2.76(dd, 1H);3.09(dd, 1H); 3.47(d, 1H); 3.72(s,3H); 3.73(s,3H); 3.99(d, 1H); 4.19(dd,1H); 4.31(m, 1H); 4.70 (m, 1H); 5.02 (dd, 1H); 6.52 (s, 1H); 6.65 (dd,1H); 7.05-7.28 (m, 4H); 7.51(d, 1H); 8.05(d, 1H); 9.74(s,1H); 9.95(s,1H) ppm

EXAMPLE 93

To a solution of 96 mg of the product of Example 51 in 1.6 ml ofmethylene chloride and 0.5 ml of dimethylformamide were added 36 mg of1-hydroxy-benzotriazole, and 42 mg ofN-(dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride. The mixturewas stirred at 0° C. for 30 minutes, then 16 mg of 2-aminoethanol wereadded, and stirring was continued at 20° C. for 1.5 hours. The mixturewas diluted with ethyl acetate and washed successively with 1Nhydrochloric acid, water, saturated sodium bicarbonate solution, andbrine. The organic layer was dried over sodium sulfate and evaporated invacuo. The residue was chromatographed on silica gel using acetone/ethylacetate (1:1, v/v) as eluent to yield, after crystallization from ethylacetate/hexane, 84 mg of(4R,7S)-7-tert-butoxycarbonylamino-1,3,4,5,6,7,8,10-octahydro-14-hydroxy-12-methoxy-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylicacid 2-hydroxyethylamide as a white solid.

¹ H-NMR(250 MHz, DMSO-d₆): δ1.41(s,9H); 1.93(s,3H); 2.70(dd, 1H);3.02(dd, 1H); 3.10(m, 2H); 3.29-3.46(m, 3H); 3.72(s,3H); 3.92(d, 1H);4.14(dd, 1H); 4.25(m, 1H); 4.48(m, 1H); 4.68(t,1H); 4.96(dd, 1H);6.48(s, 1H); 7.44(d, 1H); 7.90 (d, 1H); 8.01(t,1H); 9.70(s,1H) ppm

EXAMPLE 94

To a solution of 188 mg of the product of Example 3 in 1.6 ml ofmethylene chloride and 0.5 ml of dimethylformamide were added 68 mg of1-hydroxy-benzotriazole, and 86 mg of dicyclohexyl-carbodiimide. Themixture was stirred at 20° C. for 30 minutes, then 45 mg of morpholinewere added, and stirring was continued at 20° C. for 2 hours. Theprecipitate was removed by filtration, the filtrate was diluted withethyl acetate and washed successively with 3N hydrochloric acid, water,saturated sodium carbonate solution, and with brine. The organic layerwas dried over sodium sulfate and evaporated in vacuo. The residue waschromatographed on silica gel using ethyl acetate as eluent to yield,after trituration with ethyl acetate/hexane, 28 mg of tert-butyl (4R,7S)-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-4-morpholinocarbonyl-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamateas a white solid.

¹ H-NMR(250 MHz, DMSO-d₆): δ1.40(s,9H); 1.91(s,3H); 2.62(dd, 1H);2.85(dd, 1H); 3.28-3.66 (m, 9H); 3.93 (d, 1H); 4.14 (dd, 1H); 4.28 (m,1H); 4.80-4.98 (m, 1H); 6.45 (s, 1H); 7.26 (d, 1H); 8.08 (d, 1H); 9.45(s, 1H); 9.49 (s, 1H) ppm

EXAMPLES 95-99

Operating in an analogues manner as described in the previous example,but reacting the product of Example 3 with diethylamine, the product ofExample 51 with glycine methylester hydrochloride, sarcosine ethylesterhydrochloride, and with 2-(3-methyl-1,2,4-oxadiazol-5-yl)-ethylaminep-toluenesulfonate, and the product of Example 55 with2-(3-methyl-1,2,4-oxadiazol-5-yl)-ethylamine p-toluenesulfonate, thefollowing compounds were obtained. (If the amino component was added inthe form of its hydrochloride or 4-toluene-sulfonate salt (Examples96-99), 1 equivalent of 4-dimethylaminopyridine was added to thereaction mixture):

    __________________________________________________________________________     ##STR34##                                                                    Example                        .sup.1 H-NMR (250 MHz, DMSO-d.sub.6)           No   R.sup.2                                                                          R.sup.5         R.sup.7                                                                              δ, inter alia, ppm                       __________________________________________________________________________    95   OH CON(Et).sub.2   NHCOOBu.sup.t                                                                        0.99(t, 3H); 1.10(t, 3H); 1.38(s, 9H);                                        1.91(s, 3H); 2, 61(dd, 1H); 2.85                                              (dd, 1H); 3.10-3.40m, 4H); 3.66(d, 1H);                                       3.98(d, 1H); 4.11(dd, 1H); 4.34(m, 1H);                                       4.87(m, 1H); 4.98(dd, 1H); 6.45(s, 1H);                                       7.37(d, 1H); 7.98(d, 1H); 9.45(s, 1H);                                        9.49(s, 1H)                                    96   OMe                                                                              CONHCH.sub.2 COOMe                                                                            NHCOOBu.sup.t                                                                        1.40(s, 9H); 1.93(s, 3H); 6.67(dd, 1H)                                        3.03(dd, 1H); 3.42(d, 1H); 3.62(s, 3H);                                       3.72(s, 3H); 3.85(m, 2H); 3.92(d, 1H);                                        4.16(m, 1H); 4.30(m, 1H); 4.56(m, 1H);                                        4.94(dd, 1H); 6.50(s, 1H); 7.39(d, 1H);                                       7.95(d, 1H); 8.48(t, 1H); 9.70(s, 1H)          97   OMe                                                                               ##STR35##      NHCOOBu.sup.t                                                                        1.17(t, 3H); 1.39(s, 9H); 1.93(s, 3H);                                        3.03(s, 3H); 3.73(s, 3H); 2.91-4.16 (m,                                       3H); 4.28(m, 1H); 4.97(m, 1H); 5.57 (dd,                                      1H); 6.50(s, 1H); 7.35(d, 1H); 7.91 (d,                                       1H):9.66(s, 1H)                                98   OMe                                                                               ##STR36##      NHCOOBu.sup.t                                                                        1.41(s, 9H); 1.93(s, 3H); 2.31(s, 3H);                                        2.62(dd, 1H); 2.88-3.08(m, 3H); 3.28-                                         3.56(m, 3H); 3.72(s, 3H); 3.92(d, 1H);                                        4.15(dd, 1H); 4.27(m, 1H); 4.45(m, 1H)                                        4.95(dd, 1H); 6.51(s, 1H); 7.42(d, 1H);                                       7.91(d, 1H)8.22(t, 1H); 9.67(s, 1H)            99   OMe                                                                               ##STR37##      NHCHO  1.93(s, 3H); 2.31(s, 3H); 2.66(dd, 1H);                                       2.96-3.10(m, 3H); 3.33-3.58(m, 3H);                                           3.72(s, 3H); 3.88(d, 1H); 4.07(dd, 1H);                                       4.39(m, 1H); 4.58(m, 1H); 5.20(dd, 1H);                                       6.52(s, 1H); 7.84(d, 1H); 8.10(m, 1H);                                        8.17(s, 1H); 8.69(d, 1H); 9.73(s,              __________________________________________________________________________                                   1H)                                        

The starting material used in the Examples 98 and 99 was prepared asfollows:

(a) A solution of 0.72 g of acetamide oxime in 60 ml of ethanol wastreated with 0.6 g of a 55% dispersion of sodium hydride in mineral oil,and then stirred for 5 minutes at 20° C. To the solution were added 2.07g of methyl 3-trityamino-propanoate, and the mixture was heated atreflux for 2 hours. The solvent was evaporated in vacuo and the residuewas partitioned between ethyl acetate and 10% sodium chloride solution.The organic layer was dried over sodium sulfate, evaporated in vacuo,and the residue was chromatographed on silica gel using ethylacetate/hexane (1:5, v/v) as eluent to give 1.24 g of[2-(3-methyl-1,2,4-oxadiazol-5-yl)-ethyl]-trityl-amine as whitecrystals, m.p. 100°-101° C. (from ethyl acetate/hexane).

(b) A solution of 370 mg of[2-(3-methyl-1,2,4-oxadiazol-5-yl)-ethyl]-trityl-amine and 190 mg ofp-toluenesulfonic acid in 10 ml of ethanol was heated to 60° C. for 2hours. The solvent was evaporated and the residue was triturated withdiethylether/hexane to give 2-(3-methyl-1,2,4-oxadiazol-5-yl)ethylaminep-toluenesulfonate as white crystals of m.p. 122°-123° C.

EXAMPLE 100

A mixture of 0.71 g of tert-butyl (4R,7S)-12,14-bis(tert-butyldimethylsilyloxy)-1,3,4,5,6,7,8,10-octahydro-4-methoxycarbonyl-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamateand 0.44 g2,4-bis-(4-methoxyphenyl)-2,4-dithioxo-1,3,2,4-dithiaphosphetane in 6 mlof toluene was heated to 80° C. for 1 hour. The solvent was evaporatedin vacuo, and the residue was chromatographed on silica gel using ethylacetate/methylene chloride/hexane (1:1:1, v/v/v) as eluent. Thebis-(tert-butyldimethylsilylated)-product obtained was treated withammonium fluoride in methanol in an analogous manner to the proceduredescribed in Example 13 to yield, after crystallization fromdiethylether/hexane, 270 mg of tert-butyl (4R,7S)-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-4-methoxycarbonyl-11-methyl-10-oxo-6-thioxo-9,2,5-benzooxahiaazacyclododecine-7-carbamateas a white solid.

¹ H-NMR (250 MHz, DMSO-d₆): δ1.41 (s, 9H); 1.91 (s, 3H); 3.04 (d, 1H);3.14 (m, 1H); 3.30(m, 1H); 3.66 (s, 3H); 3.94 (d, 1H); 4.28 (dd, 1H);4.69 (m, 1H); 4.91 (m, 1H); 5.12 (m, 1H); 6.43(s,1H); 7.70(broad s, 1H);9.49(s, 1H); 9.50(s,1H); 9.55 (d, 1H) ppm

EXAMPLE 111

Using the (tert-butyldimethylsilylated)-product of Example 31 asstarting material and operating in an analogues manner as described inthe previous example, there was obtained methyl (4R)-1,3, 4, 5, 6,7,8,10-octahydro-14-hydroxy-12-methoxy-10-oxo-6-thioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylateas a white solid.

¹ H-NMR (400 MHz, DMSO-d₆): δ2.85-2.95 (m, 2H); 3.20 (dd, 1H); 3.36 (m,1H); 3.69 (s, 3H); 3.71 (s, 3H); 3.84 (d, 1H); 4.30 (d, 1H); 4.49 (m,1H); 4.69 (m, 1H); 5.40 (m, 1H); 6.59 (d, 1H); 6.77 (d, 1H); 10.06 (s,1H); 10.76 (d, 1H) ppm

EXAMPLE 102

Using the 14-(tert-butyldimethylsilylated)-product of Example 67 asstarting material and operating in an analogues manner as described inExample 100, there was obtained, after crystallization from ethylacetate/hexane, ethyl(4R,7S)1,3,4,5,6,7,8,10-octahydro-14-hydroxy-12-methoxy-11-methyl-6,10-dioxo-7-thioacetamido-9,2,5-benzoxathiaazacyclododecine-4-carboxylateas a white solid.

¹ H-NMR(250 MHz, DMSO-d₆): δ1.16 (t, 3H); 1.94 (s, 3H); 2.52 (s, 3H);2.93 (dd, 1H); 3.08 (dd, 1H); 3.64 (d, 1H); 3.72(s,3H); 3.81(d, 1H);4.07(q,2H); 4.23(dd, 1H); 4.31(m, 1H); 5.22 (dd, 1H); 5.35 (m, 1H); 6.52(s, 1H); 8.47 (d, 1H); 9.74 (s, 1H); 10.28 (d, 1H) ppm

EXAMPLE 103

As a second product from the procedure described in the previousexample, there was obtained, upon crystallization from ethylacetate/hexane, ethyl(4R,7S)-1,3,4,5,6,7,8,10-octahydro-14-hydroxy-12-methoxy-11-methyl-10-oxo-7-thioacetamido-6-thioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylate as a white solid.

¹ H-NMR (250 MHz, DMSO-d₆): 1.16 (t, 3H); 1.95 (s, 3H); 2.55 (s, 3H);3.18-3.25 (m, 2H ); 3.31 (dd, 1H); 3.72 (s, 3H); 3.94 (d, 1H); 4.08 (m,2H); 4.36 (dd, 1H); 5.01 (m, 1H); 5.23 (dd, 1H); 5.60(m, 1H);6.50(s,1H); 9.62(d, 1H); 9.76(s,1H); 10.33 (d, 1H) ppm

EXAMPLE 104

A sample of 270 mg of tert-butyl (4R,7S)-12,14-bis-(tertbutyldimethylsilyloxy)-4-carbamoyl-1,3,4,5,6,7,8,10-octahydro11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamatewas subjected in an analogues manner to the procedure described inExample 100, and the crude reaction product was treated with ammoniumfluoride in methanol in an analogous manner to the procedure describedin Example 13, to yield, after chromatographic purification on silicagel using ethyl acetate/hexane (1:1, v/v) as eluent, and crystallizationfrom ethyl acetate/hexane, 86 mg of (4R,7S)-7-(tert-butoxycarbonylamino)-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-thiocarboxamideas a white solid.

¹ H-NMR(250 MHz, DMSO-d₆): δ1.41 (s, 9H); 1.90 (s, 3H); 2.73 (dd, 1H);3.11 (dd, 1H); 3.36 (d, 1H); 3.92 (d, 1H); 4.15 (dd, 1}{); 4.19 (m, 1H);4.73 (m, 1H); 5.02 (dd, 1H); 6.45 (s, 1H); 7.60 (d, 1H); 8.00 (d, 1H);9.16 (s, 1H); 9.48 (s, 1H); 9.51 (s, 1H); 9.79 (s,1H) ppm

The starting material used above was prepared as follows:

(a) The product of Example 78 was subjected in an analogous manner tothe procedure described in Example 13 (d) to yield tert-butyl (4R,7S)-12,14-bis-(tert-butyldimethylsilyloxy)-4-carbamoyl-1,3,4,5,6,7,8,10-octahydro-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamate.

EXAMPLE 105

A solution of 28 mg of the product of Example 127 in 0.44 ml oftrifluoroacetic acid was stirred at 0° C. for 30 minutes. The solventwas evaporated in vacuo, and the residue was taken up in 2 ml of 1M pH7sodium phosphate buffer. The pH was adjusted to 8.5 by the addition of1N sodium hydroxide solution, and the solution was then chromatographedon MCI-Gel CHP20P using 0-40% aqueous acetonitrile as eluent. Theproduct-containing fractions were combined and lyophilized to give 19 mgof[(4R,7S)-7-[(N-L-alanyl-1-alanyl)-amino]-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecin-4-yl]-methylacetate as a white powder.

¹ H-NMR (250 MHz, DMSO-d₆): δ1.10(d,3H); 1.29(d, 3H); 1.88(s,3H);1.99(s,3H); 2.90(dd, 1H); 3.52 (d, 1H); 3.56-3.73 (m, 2H); 3.96-4.27 (m,6H); 4.55 (m, 1H); 5.04 (dd, 1H); 6.45(s,1H); 7.51(d, 1H); 8.14(d, 1H);9.53(broad s,2H) ppm

EXAMPLE 106

A solution of 120 mg of the 14-(tert-butyldimethylsilylated)product ofExample 21 in 2 ml of trifluoroacetic acid was stirred at 0° C. for 1hour. The solvent was evaporated in vacuo, and the residue was taken upin 4 ml of methanol. After the addition of 40 mg of ammonium fluoride,the mixture was stirred at 20° C. for 1 hour. The solvent was evaporatedin vacuo, and the residue was taken up in 2 ml of 1M pH7 sodiumphosphate buffer. The pH was adjusted to 8.5 by the addition of 1Nsodium hydroxide solution, and the solution was then chromatographed onMCI-Gel CHP20P using 0-30% aqueous acetonitrile as eluent. Theproduct-containing fractions were combined and lyophilized to give 18 mgof methyl [(4R,7S)-7-amino-1,3,4,5,6,7,8,10-octahydro-14-hydroxy-12-methoxy-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyolododecine-4-carboxylateas a white powder.

¹ H-NMR (250 MHz, DMSO-d₆): δ1.91 (S, 3H); 2.87 (dd, 1H); 3.09 (dd, 1H);3.43 (d, 1H); 3.66 (s, 3H); 3.72 s, 3H); 3.86 (d, 1H); 4.12 (dd, 1H);4.64 (m, 1H); 5.14 (dd, 1H); 6.51 (s,1H); 8.43(d, 1H); 9.70(s,1H) ppm

EXAMPLES 107-111

Using as starting material the products of Examples 78 and 94, the14-(tert-butyldimethylsilylated)-product of Example 28, and the12,14-bis-(tert-butyldimethylsilylated)-products of Examples 18 and 58,and operating in an analogues manner as described in Examples 105 and106, the following compounds were obtained:

    __________________________________________________________________________     ##STR38##                                                                    Example             .sup.1 H-NMR (250 MHz, DMSO-d.sub.6)                      No   R.sup.1                                                                          R.sup.2                                                                          R.sup.5  δ,inter alia, ppm                                   __________________________________________________________________________    107  Me OH CONH.sub.2                                                                             1.87(s, 3H); 2.73(dd, 1H); 3.03(dd, 1H);                                      3.55-3.68(m, 3H); 3.87(d, 1H); 4.11                                           (dd, 1H); 4.45(m, 2H); 5.15(dd, 1H); 6.45                                     (s, 1H); 7.23(s, 1H); 7.49(s, 1H); 8.24                                       (d, 1H); 9.52(broad s, 1H)                                108  Me OH                                                                                ##STR39##                                                                             in D.sub.2 O: δ 2.03(s, 3H); 2.85(dd, 1H);                              3.14(dd, 1H); 3.45-4.00(m, 10H); 4.40 (dd, 1H);                               5.13(dd, 1H); 5.31(dd, 1H); 6.54(s, 1H)                   109  H  OMe                                                                              COOMe    2.32(broad s, 2H); 2.86(dd, 1H); 3.05                                         dd, 1H); 3.66(s, 3H); 3.82(d, 1H); 4.17                                       (dd, 1H); 4.34(d, 1H); 4.71(m, 1H); 4.83                                      (dd, 1H); 6.49(d, 1H); 6.69(d, 1H); 8.59                                      (d, 1H); 9.64(s, 1H); 9.84(s, 1H)                         110  H  OH COOMe    2.89(dd, 1H); 3.06(dd, 1H); 3.66(s, 3H);                                      3.71(s, 3H); 3.84(d, 1H); 4.18(dd, 1H);                                       4.36(d, 1H); 4.71(m, 1H); 4.85(dd, 1H);                                       6.60(d, 1H); 6.79(d, 1H); 8.60(d, 1H);                                        10.06(broad s, 1H)                                        111  Me OH CH.sub.2 OH                                                                            1.87(s, 3H); 2.20(broad s, 2H); 2.51                                          (dd, 1H); 2.89(dd, 1H); 3.34-3.49                                             (m, 3H); 3.56(broad s, 1H); 3.78                                              (d, 1H); 3.94(m, 1H); 4.07(dd, 1H); 4.81                                      (t, 1H); 5.16(dd, 1H); 6.43(s, 1H); 7.83                                      (d, 1H); 9.44(s, 1H); 9.47(s, 1H)                         __________________________________________________________________________

EXAMPLE 112

To a solution of 1.30 g of methyl (4R,7S)-7-amino-12,14-bis(tert-butyldimethylsilyloxy)-1,3,4,5,6,7,8,10-octahydro-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylate and 0.23 g of triethylamine in 50 ml of methylene chloride,was added at 0° C. within 2 minutes a solution of 0.25 g ofchloro-acetyl chloride in 3 ml of methylene chloride. The mixture wasstirred at 0° C. for 30 minutes, then diluted with methylene chlorideand washed successively with 0.03N hydrochloric acid and water. Theorganic layer was dried over sodium sulfate and evaporated in vacuo. Theresidue was chromatographed on silica gel using ethyl acetate/hexane(1:1, v/v) as eluent to yield 1.20 g of methyl (4R, 7S)-12,14-bis(tert-butyldimethylsilyloxy)-7-(2-chloroacetamido)-1,3,4,5,6,7,8,10-octahydro-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylateas a white solid. A sample of 287 mg of this material was treated withammonium fluoride in methanol in an analogous manner to the proceduredescribed in Example 13 to yield, after crystallization from ethylacetate, 115 mg of methyl (4R,7S)-7-(2chloroacetamido)-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylate as a white solid, m.p. 182° C. (dec.).

¹ H-NMR (250 MHz, DMSO-d₆): δ2.87(dd, 1H); 3.11(dd, 1H); 3.63(S,3H);3.90(d, 1H); 4.15-4.33(m, 2H) superimposed by 4.23 (s, 2H); 4.50-4.68(m, 2H); 4.87 (dd, 1H); 6.52 (dd, 1H); 6.73 (dd, 1H); 8.53 (d, 1H); 8.70(d, 1H); 9.63 (s, 1H); 9.83 (s, 1H) ppm

The starting material used above was prepared as follows:

(a) tert-Butyl (4R, 7S)-12, 14-bis(tert-butyldimethylsilyloxy)-1,3,4,5,6,7,8,10-octahydro-4-methoxycarbonyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamatewas subjected in an analogous manner to the procedure described inExample 14 (a) to give methyl (4R, 7S)-7-amino-12, 14-bis(tert-butyldimethylsilyloxy)-1,3,4,5,6,7,8,10-octahydro-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylate.

EXAMPLE 113

A solution of 45 mg of methyl (4R,7S)-7-(2-chloroacetamido)-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylatein 0.5 ml of morpholine was stirred at 20° C. for 15 minutes. Thesolution was evaporated in vacuo, and to the residue were added 5 ml of0.1N hydrochloric acid, a white precipitate forming immediately. Theprecipitate was isolated by centrifugation, washed with water and driedto yield 33 mg of methyl 4-[(4R, 7S)-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-4-methoxycarbonyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecin-7-yl-carbamoyl-methyl]-morpholinehydrochloride as a white solid.

¹ H-NMR(250 MHz,DMSO-d₆): δ2.45 (m, 4H); 2.93 (dd, 1H); 3.02 (d, 1H);3.10 (d, 1H); 3.14 (dd, 1H); 3.50(m, 1H); 3.63(s,3H); 3.88(d, 1H);4.09(d, 1H); 4.29(dd, 1H); 4.48 (m, 1H); 4.68 (m, 1H); 4.86 (dd, 1H);6.50 (d, 1H); 6.63 (d, 1H); 8.25 (d, 1H); 8.45(d, 1H); 9.64(s,1H);9.85(s,1H) ppm

EXAMPLE 114-116

By operating in an analogues manner as described in the previousexample, but replacing morpholine by 1-methylpiperidine, pyridine or by1,4-diazabicyclo[2.2.21octane, the following compounds were obtained.

    __________________________________________________________________________     ##STR40##                                                                    Example               .sup.1 H-NMR (250 MHz, DMSO-d.sub.6)                    No   R7               δ, inter alia, ppm                                __________________________________________________________________________    114                                                                                 ##STR41##       2.65(s, 3H); 2.94(dd, 1H); 3.15(dd, 1H); 3.20 (s,                             2H); 3.63(s, 3H); 3.89(d, 1H); 4.16 (d, 1H);                                  4.26(dd, 1H); 4.49(m, 1H); 4.68 (m, 1H); 4.87(m,                              1H); 6.56(d, 1H); 6.67 (d, 1H); 8.34(d, 1H);                                  8.54(d, 1H); 9.70 (s, 1H); 9.93(s, 1H); 10.41                                 (broad s, 1H)                                           115                                                                                 ##STR42##       3.03(dd, 1H); 3.16(dd, 1H); 3.61(s, 3H); 4.00 (d,                             1H); 4.25(dd, 1H); 4.40(d, 1H); 4.50-4.67 (m, 2H);                            4.94(dd, 1H); 5.56(d, 1H); 5.80  (d, 1H); 6.60(d,                             1H); 6.75(d, 1H); 8.22 (t, 2H); 8.68(t, 1H);                                  8.87(d, 1H); 9.08 (d, 2H); 9.39(d, 1H); 9.73(s,                               1H); 9.97(s, 1H)                                        116                                                                                 ##STR43##       2.90-3.16(m, 8H); 3.39-3.60(m, 6H); 3.61 (s, 3H);                             3.95(d, 1H); 4.12-4.30(m, 3H); 4.37 (d, 1H);                                  4.61(m, 1H); 4.71(m, 1H); 4.94 (dd, 1H); 6.60(d,                              1H); 6.73(d, 1H); 8.75 (d, 1H); 9.33(d, 1H);                                  9.73(s, 1H); 9.98(s, 1H)                                __________________________________________________________________________

EXAMPLE 117

A solution of 51 mg of methyl (4R,7S)-7-(2-chloroacetamido)-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylate,18 mg of tetrahydro-2-methyl-3-thioxo-as-triazine-5,6-dione, and 12 mgof triethylamine in 1 ml of dimethylformamide was stirred for 4 days at20° C. The solvent was evaporated in vacuo, and the residue was taken upin 20% aqueous acetonitrile and chromatographed on MCI-Gel CHP20P using0-30% aqueous acetonitrile as eluent. The product-containing fractionswere combined and lyophilized to give 32 mg of methyl (4R,7S)-7-[2-[((2,5-dihydro-6-hydroxy-2-methyl-5-oxo-as-triazin-3-yl)thio]acetamido]-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylateas a white solid.

¹ H-NMR(250 MHz, DMSO-d₆): δ3.09 (m, 2H); 3.53 (s, 3H); 3.64 (s, 3H);3.94 (d, 1H): 4.04 (d, 1H); 4.10 (d, 1H); 4.20(d, 1H); 4.47(d, 1H);4.57(m, 1H); 4.76(m, 2H); 4.87 (dd, 1H); 6.54 (d, 1H); 6.80 (d, 1H);7.84 (d, 1H); 9.07 (d, 1H); 9.64 (s, 1H); 9.84 (s, 1H) ppm

EXAMPLE 118

To a mixture of 245 mg of methyl (4R, 7S)-7-amino-12,14-bis(tert-butyldimethylsilyloxy)-11-methyl-1,3,4,5,6,7,8,10-octahydro-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylateand 102 mg of 3[N-(tert-butoxycarbonyl)methylamino]-propionic acid in 6ml of acetonitrile, cooled to 0° C., were added 95 mg ofN-(dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride. The mixturewas stirred at 0° C. for 3 hours, and then diluted with ethyl acetateand washed successively with 3N hydrochloric acid, water, saturatedsodium carbonate solution, and brine. The organic layer was dried oversodium sulfate and evaporated in vacuo. The residue was dissolved in 5ml of cold trifluoroacetic acid, and the solution was stirred at 0° C.for 30 minutes. The solvent was evaporated in vacuo, the residue wastaken up in ethyl acetate, and the solution was washed with saturatedsodium bicarbonate solution, and with brine. The organic layer was driedover sodium sulfate end evaporated in vacuo. The residue was treatedwith ammonium fluoride in methanol in an analogous manner to theprocedure described in Example 13. The resulting product was purified bychromatography on MCI-Gel CHP20P which had been pretreated with 1%aqueous acetic acid. Using 0-20% aqueous acetonitrile as eluent, therewere obtained, alter lyophilization of the product-containing fractions,49 mg of methyl (4R,7S)-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-11-methyl-7-[3-(methylamino)propionamido]-6,10-dioxo-9,2,5-benzooxathiaazacyclododecine-4-carboxylate acetate as a white solid.

¹ H-NMR (250 MHz,DMSO-d₆): δ1.89 (s, 3H), 2.24 (s, 3H); 2.66-2.82 (m,3H); 3.09 (dd, 1H); 3.55 (d,1H); 3.63(s,3H); 3.86(d,1H); 4.05(dd,1H)4.62(m, 1H); 5.35 (dd, 1H); 6.46 (s, 1H); 8.51 (d, 1H); 8.65 (d, 1H)ppm

EXAMPLE 119

A solution of 63 mg of methyl(4R,7S)-7-amino-14-(tert-butyldimethylsilyloxy)-1,3,4,5,6,7,8,10-octahydro-12-methoxy-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylateand 20 ml of pyridine in 2 ml of acetic anhydride was heated to 60° C.for 1 hour. The solution was evaporated in vacuo, the residue wasdissolved in ethyl acetate, and the solution was washed with saturatedsodium bicarbonate solution, and with brine. The organic layer was driedover sodium sulfate and evaporated in vacuo. The residue was treatedwith ammonium fluoride in methanol in an analogous manner to theprocedure described in Example 13 to yield, after crystallization frommethanol/ethyl acetate/hecate, 30 mg of methyl (4R,7S)-7-acetylamino-1,3,4,5,6,7,8,10-octahydro-14-hydroxy-12-methoxy-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylateas a white solid.

¹ H-NMR (250 MHz, DMSO-d₆): δ1.94 (s, 6H): 2.87 (dd, 1H); 3.08 (dd, 1H);3.54 (d, 1H); 3.63 (s, 3H); 3.72 (s,3H); 3.80(d, 1H); 4.11(dd,1H);4.45(m, 1H); 4.67(m, 1H); 5.10 (dd, 1H); 6.51 (s,1H); 8.27(d, 1H); 9.73(s, 1H)ppm

The starting material used above was prepared as follows:

(a) The 12,14-bis (tert-butyldimethylsilylated)-product of Example 21was subjected in an analogous manner to the procedure described inExample 14 (a) to give methyl (4R,7S)-7-amino-14-(tert-butyldimethylsilyloxy)-1,3,4,5,6,7,8,10-octahydro-12-methoxy-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylate.

EXAMPLE 120

A sample of 156 mg of the product of Example 14(a) was subjected in ananalogues manner to the procedure described in Example 119 to give,after crystallization from acetone/hexane, 79 mg of methyl(4R,7S)-7-acetylamino-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylateas a white solid.

¹ H-NMR(250 MHz, DMSO-d₆): δ1.90(s,3H); 1.94(s,3H); 2.89(dd, 1H);3.10(dd, 1H); 3.50(d, 1H); 3.63 (s, 3H); 3.77 (d, 1H); 4.09 (dd, 1H);4.45 (m, 1H); 4.67 (m, 1H); 5.10 (dd, 1H); 6.45(s,1H); 8.26(d, 1H);8.29(d, 1H); 9.49(s,1H); 9.51(s,1H) ppm

EXAMPLE 121

The 12,14-bis(tert-butyldimethylsilylated)-Product of Example 108 wassubjected in an analogues manner to the procedure described in Example119 to give N-[(4R,7S)-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-11-methyl-4-(morpholinocarbonyl)-6,10-dioxo-9,2,5-benzoxathiaazacyclododecin-7-yl]acetamideas a white solid.

¹ H-NMR (250 MHz, DMSO-d₆): δ1.91(s,3H); 1.94(s,3H); 2.75(dd, 1H);2.89(dd, 1H); 3.22-3.70 (m, ˜5H); 3.96 (d, 1H); 4.03 (dd, 1H); 4.55 (m,1H); 4.87 (m, 1H); 5.17 (dd, 1H); 6.48 (s, 1H); 8.02 (d, 1H); 8.28 (d,1H); 9.57 (broad s, 1H) ppm

EXAMPLE 122

To a mixture of 61 mg of methyl(4R,7S)-7-amino-12,14-bis-(tert-butyldimethylsilyloxy)-11-methyl-1,3,4,5,6,7,8,10-octahydro-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylateand 16 mg of 3-methoxypropionic acid in 2 ml of acetonitrile, cooled to0° C., were added 29 mg of N-(dimethylaminopropyl)-N'-ethylcarbodiimidehydrochloride. The mixture was stirred at 0° C. for 3 hours, and thendiluted with ethyl acetate and washed successively with 3N hydrochloricacid, water, 5% sodium bicarbonate solution, and with brine. The organiclayer was dried over sodium sulfate and evaporated in vacuo. The residuewas chromatographed on silica gel using ethyl acetate/hexane (1:1, v/v)as eluent, and the (tert-butyldimethylsilylated)-product obtained wastreated with ammonium fluoride in methanol in an analogous manner to theprocedure described in Example 13 to yield, after crystallization fromethyl acetate/hexane, 24 mg of methyl (4R,7S)-7-(3-methoxy-propionylamino)-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclo-dodecine-4-carboxylateas a white solid.

¹ H-NMR(250 MHz, DMSO-d₆): δ1.90 (s, 3H); 2.48 {m, 2H ); 2.84(dd, 1H);3.09(dd, 1H); 3.21(s,3H); 3.49 (d, 1H); 3.55 (t, 1H); 3.62(s,3H);3.76(d, 1H); 4.08(dd, 1H); 4.44 (m, 1H); 4.67 (m, 1H); 5.07 (dd, 1H);6.45 (s, 1H); 8.14 (d, 1H); 8.32 (d, 1H); 9.45 (s, 1H); 9.52 (s, 1H) ppm

EXAMPLE 123-129

Operating in an analogues manner as described in the previous example,the product of Example 14 (a) was acylated withtrans-N-(tert-butoxycarbonyl)-4-hydroxy-L-proline,N-(tert-butoxycarbonyl)-L-threonine, 3-cyclopentyl-propionic acid, andwith 3-phenyl-propionic=acid, the12,14-bis-(tert-butyldimethylsilylated)-product of Example 64 was firsttreated with trifluoroacetic acid in an analogues manner as described inExample 105 and the resulting product was acylated withN-(tert-butoxycarbonyl)-L-alanyl-L-alanine, the product of Example119(a) was acylated with cyclopropane-carboxylic acid and with3,4-dihydroxycinnamic acid, yield the following compounds:

    __________________________________________________________________________     ##STR44##                                                                    Example                         .sup.1 H-NMR (250 MHz, DMSO-d.sub.6)          No   R2 R5   R7                 δ, inter alia, ppm                      __________________________________________________________________________    123  OH COOMe                                                                               ##STR45##         1.31(s, 9H); 1.78-2.00(m, 1H) superim-                                        posed by 1.90(s, 3H); 2.00-2.20 (m, 1H);                                      2.80-3.14(m, 2H); 3.30-3.50 (m, 3H);                                          3.62(s, 3H); 3.80(d, 1H); 4.12 (m, 1H);                                       4.24(m, 1H); 4.33(m, 2H); 4.68 (m, 1H);                                       4.95(m, 1H); 5.02(d, 1H); 6.45 (s, 1H);                                       8.20(d, 1H); 8.52(d, 1H); 9.51 (s, 1H);                                       9.53(s, 1H)                                   124  OH COOMe                                                                               ##STR46##         1.04(d, 3H); 1.37(s, 9H); 1.90(s, 3H);                                        2.72(dd, 1H); 3.12(dd, 1H); 3.48(d, 1H);                                      3.60(s, 3H); 3.73(d, 1H); 4.00(m, 1H);                                        4.11(dd, 1H); 4.21(m, 1H); 4.45(m, 1H);                                       4.68(m, 1H); 5.13(d, 1H); 5.22(m, 1H);                                        6.47(s, 1H); 6.54(d, 1H); 8.21(d, 1H);                                        8.34(d, 1H), 9.54(s, 1H); 9.56(s, 1H)         125  OH COOMe                                                                               ##STR47##         0.96-1.16(m, 2H); 1.40-1.80(m, 10H);                                          1.90(s, 3H); 2.24(m, 2H); 2.87(dd, 1H);                                       3.06(dd, 1H); 3.47(d, 1H); 3.62(s, 3H);                                       3.77(d, 1H); 4.10(dd, 1H); 4.40(m, 1H);                                       4.64(m, 1H); 5.00(dd, 1H); 6.45(s, 1H);                                       8.18(d, 1H); 8.24(d, 1H); 9.50(s, 1H)                                         9.52(s, 1H)                                   126  OH COOMe                                                                               ##STR48##         1.90(s, 3H); 2.53(m, 2H); 2.75-2.95 (m,                                       3H); 3.08(dd, 1H); 3.48(d, 1H); 3.62 (s,                                      3H); 3.74(d, 1H); 4.03(dd, 1H); 4.38 (m,                                      1H); 4.65(m, 1H); 5.01(dd, 1H); 6.45 (s,                                      1H); 7.10-7.30(m, 5H); 8.23(d, 1H)                                            8.30(d, 1H); 9.50(s, 1H); 9.52(s, 1H)         127  OH CH.sub.2 OAc                                                                        ##STR49##         1.12(d, 3H); 1.26(d, 3H); 1.35(s, 9H);                                        1.89(s, 3H); 1.99(s, 3H); 2.90(dd, 1H);                                       3.49(d, 1H); 3.74(d, 1H); 3.88-4.06 (m,                                       4H); 4.14(dd, 1H); 4.26(m, 1H); 4.57 (m,                                      1H); 5.05(dd, 1H); 6.45(s, 1H); 6.91 (d,                                      1H); 7.57(d, 1H); 8.06(d, 1H); 8.14 (d,                                       1H); 9.49(s, 1H); 9.51(s, 1H)                 128  OMe                                                                              COOMe                                                                               ##STR50##         0.70(m, 4H); 1.83(m, 1H); 1.94(s, 3H);                                        2.93(dd, 1H); 3.10(dd, 1H); 3.57(d, 1H)                                       3.63(s, 3H); s, 72(s, 3H); 3.78(d, 1H);                                       4.15(dd, 1H); 4.35(m, 1H); 4.71(m, 1H);                                       5.00(dd, 1H); 6.52(s, 1H); 8.32(d, 1H);                                       8.56(d, 1H); 9.75(s, 1H)                      129  OMe                                                                              COOMe                                                                               ##STR51##         1.94(s, 3H); 2.90(dd, 1H); 3.09(dd, 1H);                                      3.56(d, 1H); 3.62(s, 3H); 3.73(s, 3H);                                        3.82(d, 1H); 4.45(m, 1H); 4.79(m, 1H);                                        5.17(dd, 1H); 6.53(s, 1H); 6.64(d, 1H);                                       6.74(d, 1H); 6.88(dd, 1H); 6.97(d, 1H);                                       7.30(d, 1H); 8.35(d, 1H); 8.39(d, 1H);                                        9.15(s, 1H); 9.43(s, 1H); 9.75(s,             __________________________________________________________________________                                    1H)                                       

EXAMPLE 130

A solution of 52 mg of methyl (4R,7S)-7-amino-14-(tert-butyldimethylsilyloxy)-1,3,4,5,6,7,8,10-octahydro-12-methoxy-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylateand 100 mg of 4-nitrophenyl formate in 2 ml of methylene chloride washeated at reflux for 18 hours. The solution was evaporated in vacuo, andthe residue was chromatographed on silica gel using ethyl acetate/hexane(2:1, v/v) as eluent. The (tert-butyldimethylsilylated)-product obtainedwas treated with ammonium fluoride in methanol in an analogous manner tothe procedure described in Example 13 to yield, after crystallizationfrom acetone/hexane, 20 mg of methyl (4R,7S)-7-formylamino-1,3,4,5,6,7,8,10-octahydro-14-hydroxy-12-methoxy-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylateas a white solid.

¹ H-NMR (250MHZ, DMSO-d₆): δ1.93(s,3H); 2.86(dd, 1H); 3.10(dd, 1H);3.57(d, 1H); 3.63(S,3H); 3.72 (s,3H); 3.76(d, 1H); 4,12(dd, 1H); 4.44(m,1H); 4.72(m, 1H); 5.12 (dd, 1H); 6.52(s, 1H); 8.14 (s, 1H); 8.40(d, 1H);8.58(d, 1H); 9.74 (s, 1H) ppm

EXAMPLE 13 1

Methyl (4R,7S)-7-amino-14-(tert-butyldimethylsilyloxy)-1,3,4,5,6,7,8,10-octahydro-12-methoxy-6,10-dioxo-9,2,5-benzoxathiaazacyciododecine-4-carboxylatewas acylated with allylacetic acid in an analogues manner as describedin Example 122 and the protection groups were subsequently cleaved offusing the procedure described in Example 13 to yield methyl(4R,7S)-7-(pent-4-enoylamino)-1,3,4,5,6,7,8,10-octahydro-14-hydroxy-12-methoxy-6,10-dioxo-9,2,5-benzooxathiaazacyclododecine-4-carboxylateas a white solid.

¹ H-NMR (250 MHz,CDCl₃): δ2.30-2.50 (m, 4H); 3.11 (dd, 1H); 3.21 (dd,1H); 3.77 (s, 3H); 3.80 (s,3H); 3.94(d, 1H); 4.14 (d, 1H); 4.56(dd, 1H);4.82-5.22(m, ˜5H); 5.50-6.00 (m, ˜2H); 6.63 (d, 1H); 6.79 (d, 1H); 6.91(d, 1H); 7.17 (d, 1H) ppm

The starting material used above was prepared as follows:

(a) tert-Butyl (4R,7S)-14-(tert-butyldimethylsilyloxy)-1,3,4,5,6,7,8,10-octahydro-12-methoxy-4-methoxycarbonyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamatewas subjected in an analogous manner to the procedure described inExample 14 (a) to give methyl (4R,7S)-7-amino-14-(tert-butyldimethylsilyloxy)-1,3,4,5,6,7,8,10-octahydro-12-methoxy-6,10-dioxo9,2,5-benzoxathiaazacyclododecine-4-carboxylate.

EXAMPLE 132

To a solution of 51 mg of the product of Example 119(a) in 0.7 ml ofmethylene chloride were added at 0° C. 14 mg of propionyl chloride, andof 18 mg 4-dimethylamino-pyridine. The mixture was stirred at 0° C. for1.5 hours, then diluted with ethyl acetate and washed successively with3N hydrochloric acid, water, 5% sodium bicarbonate solution, and withbrine. The organic layer was dried over sodium sulfate and evaporated invacuo. The residue was chromatographed on silica gel using ethylacetate/hexane (1:1, v/v) as eluent, and the(tert-butyldimethylsilylated)-product obtained was treated with ammoniumfluoride in methanol in an analogous manner to the procedure describedin Example 13 to yield, after crystallization from ethyl acetate/hexane,10 mg of methyl (4R,7S)-1,3,4,5,6,7,8,10-octahydro-14-hydroxy-12-methoxy-11-methyl-6,10-dioxo-7-propionylamino-9,2,5-benzoxathiaazacyclododecine-4-carboxylate as awhite solid.

¹ H-NMR(250 MHz, DMSO-d₆): δ1.01 (t, 3H); 1.93 (s, 3H); 2.24 (q, 2H);2.88 (dd, 1H); 3.08 (dd, 1H); 3.50 (d, 1H); 3.62 (s, 3H); 3.72 (s, 3H);3.78 (d, 1H); 4.12 (dd, 1H); 4.64 (m, 1H); 5.03 (dd, 1H); 6.51 (s, 1H);8.18 (d, 1H); 8.21 (d, 1H); 9.75 (s, 1H) ppm

EXAMPLES 133-141

Operating in an analogues manner as described in the previous example,the product of Example 14 (a) was acylated with 3,4,5-trimethoxy-benzoylchloride and with methanesulfonyl chloride, the product of Example 112(a) was acylated with 2,3-dioxosulfinyl-benzoyl chloride, the product ofexample 119(a) was acylated with pyridine-3-carboxylic acid chloride andwith methyl chloroformate, the product of Example 131 (a) was acylatedwith oxalylic acid mono-methylester chloride and withpyridine-3-sulfonyl chloride, and methyl (4R,7S)-14-(tert-butyldimethylsilyloxy)-1,3,4,5,6,7,8,10-octahydro-12-methoxy-11-methyl-7-methylamino-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylatewas acylated with acetyl chloride and with benzoyl chloride, to yieldthe following compounds:

    __________________________________________________________________________     ##STR52##                                                                    Example                   .sup.1 H-NMR (250 MHz, DMSO-d.sub.6)                No   R.sup.1                                                                          R.sup.2                                                                          R.sup.7        δ, inter alia, ppm                            __________________________________________________________________________    133  Me OH                                                                                ##STR53##     1.92(s, 3H); 2.86(dd, 1H); 3.10 (dd, 1H);                                     3.60(s, 3H); 3.61(d, 1H); 3.70 (d, 1H); 3.71(s,                               3H); 3.84(s, 6H); 4.38- 4.53(m, 2H);                                          4.85-4.99(m, 2H); 6.46 (s, 1H); 7.21(s, 2H);                                  8.49(d, 1H); 8.57(d, 1H); 9.51(s, 1H); 9.54(s,                                1H)                                                 134  Me OH NHSO2Me        1.99(s, 3H); 2.90(dd, 1H); 3.01(s, 1H);                                       3.10(dd, 1H); 3.49(d, 1H); 3.63(s, 3H);                                       3.70(d, 1H); 4.20-4.50(m, 2H); 4.93                                           (dd, 1H); 6.48(s, 1H); 7.86(d, 1H); 8.49                                      (d, 1H); 9.50(s, 1H); 9.52(s, 1H)                   135  H  OH                                                                                ##STR54##     2.91(dd, 1H); 3.07(dd, 1H); 3.61 (s, 3H);                                     3.92(d, 1H); 4.33(dd, 1H); 4.37 (d, 1H);                                      4.63(m, 1H); 4.83(m, 1H); 4.97 (dd, 1H);                                      6.47(d, 1H); 6.53(d, 1H); 6.75 (d, 1H);                                       6.80(dd, 1H); 7.30(dd, 1H); 8.47(d, 1H)             136  Me OMe                                                                               ##STR55##     1.96(s, 3H); 2.82(dd, 1H); 3.08 (dd, 1H);                                     3.58(d, 1H); 3.60(s, 3H); 3.73 (s, 3H); 3.82(d,                               1H); 4.37(dd, 1H); 4.52 (m, 1H); 4.88(m, 1H);                                 5.06(dd, 1H); 6.53 (s, 1H); 7.55(dd, 1H);                                     8.24(m, 1H); 8.47 (d, 1H); 8.75(dd, 1H);                                      8.88(d, 1H); 9.05 (d, 1H); 9.75(broad s, 1H)        137  Me OMe                                                                              NHCOOMe        1.92(3.3H), 2.83(dd, 1H); 3.07                                                (dd, 1H); 3.54(d, 1H); 3.60(s, 3H); 3.63                                      (s, 3H); 3.72(s, 3H); 3.79(d, 1H); 4.21                                       (dd, 1H); 4.37-4.54(m, 2H); 4.99                                              (dd, 1H); 6.51(s, 1H); 7.51(d, 1H); 8.33                                      (d, 1H); 9.71(s, 1H)                                138  H  OMe                                                                              NHCOCOOMe      2.85(dd, 1H); 3.12(dd, 1H); 3.64                                              (s, 3H); 3.72(s, 3H); 3.81(s, 1H); 3.88                                       (d, 1H); 4.23(d, 1H); 4.37(dd, 1H);                                           4.50-4.75(m, 2H); 4.86(dd, 1H);                                               6.62(d, 1H); 6.82(d, 1H); 8.65(d, 1H);                                        8.93(d, 1H):10.05(s, 1H)                            139  H  OMe                                                                               ##STR56##     2.86-3.16,(m, 2H); 3.63(s, 3H); 3.70 (s, 3H);                                 3.89(d, 1H); 4.16-4.33(m, 2H); 4.49-4.75(m,                                   2H); 6.60(d, 1H); 6.79 (d, 1H).; 7.73(m, 1H);                                 8.26(m, 1H); 8.63 (d, 1H); 8.84(m, 2H); 9.02(d,                               1H); 10.07 (s, 1H)                                  140  Me OMe                                                                              N(Me)COMe      1.90(s, 3H); 2.08(s, 3H); 2.73(dd, 1H);                                       3.12(s, 3H); 3.18(dd, 1H); 3.64(s, 3H);                                       3.72(s, 3H); 4.24(dd, 1H); 4.40-4.74                                          (m, 2H); 5.39(m, 1H); 6.51(s, 1H); 8.29                                       (d, 1H); 9.71(s, 1H)                                141  Me OMe                                                                               ##STR57##     1.90(s, 3H); 2.76(m, 1H); 3.00-3.25 (m, 4H);                                  3.60-3.80(m, 5H) superim-  posed by 3.72(s,                                   3H); 4.18-4.70 (m, 3H); 5.46(m, 1H); 6.51(s,                                  1H); 7.42 (s, 5H); 9.70(s, 1H)                      __________________________________________________________________________

EXAMPLE 142

A sample of 59 mg of the product of Example 119(a) was acylated with 60mg of 3-trityloxypropionyl chloride in an analogues manner to theproceduree described in Example 132. To a suspension of the14-(tert-butyldimethylsilylated)-product obtained in 2 ml of methanolwere added 8 mg of p-toluenesulfonic acid mono-hydrate, and the mixturewas stirred at 50° C. for 8 minutes. The solvent was evaporated, and theresidue was chromatographed on silica gel using ethyl acetate as eluent.The 14-(tert-butyldimethylsilylated)-product obtained was treated withammonium fluoride in methanol in an analogous manner to the proceduredescribed in Example 13 to yield, after crystallization frommethanol/diethylether, 10 mg of methyl (4R,7S)-1,3,4,5,6,7,8,10-octahydro-14-hydroxy-7-(3-hydroxy-propionylamino)-12-methoxy-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylateas a white solid.

¹ H-NMR (250 MHz, DMSO-d₆): δ1.93 (s, 3H); 2.30-2.50 (m, 4H); 2.82 (dd,1H); 3.08 (dd, 1H); 3.52 (d, 1H); 3.60-3.78 (m, 2H) superimposed by 3.63(s, 3H) and 3.72 (s, 3H); 3.80 (d, 1H); 4.10 (dd, 1H); 4.44 (m, 1H);4.64 (m, 1H); 4.76 (t, 1H); 5.13(dd, 1H); 6.52(s, 1H); 8.23(d, 1H);8.28(d, 1H); 9.75(s, 1H) ppm

EXAMPLE 143

The product of Example 14(a) was acylated with2,2-diphenyl-1,3-benzodioxole-5-sulfonyl chloride in an analogues mannerthe procedure described in Example 132. The resulting product wasstirred in 80% aqueous trifluoroacetic acid, and then the mixture wasevaporated in vacuo. The residue was stirred with diethylether/hexaneand the crystals formed were isolated by filtration to yield methyl (4R,7S)-12,14-dihydroxy-7-(3,4-dihydroxybenzenesulfonamido)-1,3,4,5,6,7,8,10-octahydro-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylate.

¹ H-NMR (250 MHz, DMSO-d₆): δ1.86 (s, 3H); 2.84 (dd, 1H); 3.03 (dd, 1H);3.39 (d, 1H); 3.62 (s, 3H); 3.79(d, 1H); 3.81(m, 1H); 3.92(m, 1H);4.42(m, 1H); 4.88(dd, 1H); 6.46 (s, 1H); 6.87 (d, 1H); 7.38 (dd, 1H);7.43 (d, 1H); 8.39 (d, 1H); 8.44 (d, 1H); 9.50(s,1H); 9.52(s,1H);9.63(s,1H); 9.94(s, 1H) ppm

EXAMPLE 144

A solution of 28 mg of the product of Example 131(a) and 5.2 mg ofmethylisothiocyanate in 2 ml of tetrahydrofuran is heated to 60° C. for3 hours. The solvent was evaporated in vacuo and the residue wascrystallized from diethylether/pentane to yield 25 mg of methyl (4R,7S)-14-(tertbutyldimethylsilyloxy)-1,3,4,5,6,7,8,10-octahydro-12-methoxy7-(3-methyl-thioureido)-6,10-dioxo-9,2,5-benzoxathiaazacyclo-dodecine-4-carboxylate.

A sample of 22 mg of this material was treated with ammonium fluoride inmethanol in an analogous manner to the procedure described in Example 13to yield, after crystallization from ethyl acetate/pentane, 15 mg ofmethyl (4R, 7S)-1,3,4,5,6,7,8,10-octahydro-14-hydroxy-12-methoxy-7-(3-methyl-thioureido)-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylateas a white solid.

¹ H-NMR(250 MHz, DMSO-d₆): δ2.89(d, 3H); 2.99(dd, 1H); 3.11(dd, 1H);3.63(s,3H); 3.71(s,3H); 3.92 (d, 1H); 4.18 (d, 1H); 4.25 (m, 1H); 4.41(m, 1H); 5.00 (m, 1H); 5.23 (m, 1H); 6.58 (d, 1H); 6.75 (d, 1H); 7.75(m, 1H); 7.88 (m, 1H); 8.62 (d, 1H); 10.06(s, 1H) ppm

EXAMPLES 145-149

By the reaction of the product of Example 131 (a) withbenzoylisothiocyanate and with benzoyl-isocyanate in an analogous manneras described in the previous example, and by the reaction of the productof Example 112 (a) with 4-bromophenylisocyanate in refluxingacetonitrile, and with potassium cyanate in acetic acid at 20° C. withsubsequent cleavage of the (tert-butyldimethylsilyl)-groups using theprocedure described in Example 13, and by the reaction of the product ofExample 110 with 4-chlorophenyl-isothiocyanate, the following compoundswere obtained:

    __________________________________________________________________________     ##STR58##                                                                    Example                 .sup.1 H-NMR (250 MHz, DMSO-d.sub.6)                  No   R.sup.2                                                                          R.sup.7         δ, inter alia, ppm                              __________________________________________________________________________    145  OMe                                                                               ##STR59##      2.98(dd, 1H); 3.14(dd, 1H); 3.64 (s, 3H); 3.71(s,                             3H); 3.96(d, 1H); 4.16 (d, 1H); 4.38-4.60(m, 2H);                             5.19 (dd, 1H); 5.27(m, 1H); 6.60(d, 1H); 6.74 (d,                             1H); 7.48(t, 2H); 7.63(t, 1H); 7.93 (d, 1H);                                  8.94(d, 1H); 10.11(s, 1H); 11.69(s, 1H)               146  OMe                                                                               ##STR60##      2.92(dd, 1H); 3.02(dd, 1H); 3.64 (s, 3H); 3.72(s,                             3H); 3.93(d, 1H); 4.21 (d, 1H); 4.34(dd, 1H);                                 4.58(m, 1H); 4.74 (m, 1H); 5.13(dd, 1H); 6.60(d,                              1H); 6.74 (d, 1H); 7.51(m, 2H); 7.63(m, 1H); 8.00                             m, 2H); 8.83(d, 1H); 9.50(d, 1H); 10.14 (s, 1H);                              11.02(s, 1H)                                          147  OH                                                                                ##STR61##      2.96(dd, 1H); 3.14(dd, 1H); 3.63 (s, 3H); 3.92(d,                             1H); 4.23(d, 1H); 4.50- 4.64(m, 2H); 4.99(dd,                                 1H); 6.51 (d, 1H); 6.67(d, 1H); 6.79(d, 1H); 7.40                             m, 4H); 8.64(d, 1H); 9.00(s, 1H); 9.65 (s, 1H)                                9.85(s, 1H)                                           148  OH NHCONH2         2.95(dd, 1H); 3.09(dd, 1H); 3.64                                              (s, 1H); 3.89(d, 1H); 4.14(dd, 1H); 4.22                                      (d, 1H); 3.48(m, 1H); 3.53(m, 1H); 3.90                                       (dd, 1H); 5.87(s, 2H); 6.49(d, 1H); 6.56                                      (d, 1H); 6.67(d, 1H); 8.39(d, 1H); 9.63                                       (s, 1H); 9.83(s, 1H)                                  149  OH                                                                                ##STR62##      3.02(dd, 1H); 3.15(dd, 1H); 3.64 (s, 3H); 3.94(d,                             1H); 4.21(d, 1H); 4.37 (dd, 1H); 4.44(m, 1H);                                 5.07(dd, 1H); 5.29 (m, 1H); 6.51(d, 1H); 6.66(d,                              1H); 7.37 (d, 2H); 7.67(d, 2H); 8.16(d, 1H); 8.79                             d, 1H); 9.66(s, 1H); 9.86(s, 1H); 10.15 (s,           __________________________________________________________________________                            1H)                                               

EXAMPLE 150

To a solution of 184 mg of the product of Example 14 (a), 240 mg ofacetone, and 49 mg of sodium acetate in 1 ml of 25% aqueous acetic acid,cooled to 0° C., were added portionwise within 1 hour 80 mg of sodiumborohydride. The mixture was diluted with ethyl acetate and washed withsaturated sodium carbonate solution and with brine. The organic layerwas dried over sodium sulfate and evaporated in vacuo. The residue waschromatographed on silica gel using ethyl acetate/hexane (1:3, v/v) togive, after crystallization from diethylether/hexane, 54 mg12,14-bis(tert-butyldimethylsilylated)-product. A sample of 34 mg ofthis material was treated with ammonium fluoride in methanol in ananalogous manner to the procedure described in Example 13 to yield,after crystallization from methylene chloride/hexane, 15 mg of methyl(4R,7S)-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-7-(isopropylamino)-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylateas a white solid.

¹ H-NMR(250 MHz, DMSO-d₆): δ1.01(d, 3H); 1.04(d, 3H); 1.88(s,3H);2.91(dd, 1H); 3.07(dd, 1H); 3.38 (d, 1H); 3.45 (m, 1H); 3.64 (s, 3H);3.80 (d, 1H); 4.23 (dd, 1H); 4.55 (m, 1H); 4.88 (dd, 1H); 6.44 (s, 1H);8.49 (d, 1H); 9.48 (s, 1H); 9,50 (s, 1H) ppm

EXAMPLE 151

Operating in an analogues manner as described in the previous example,but replacing acetone by p-hydroxybenzaldehyde, there was obtainedmethyl (4R,7S)-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-7-[(p-hydroxybenzyl)amino]-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylateas a white solid.

¹ H-NMR (250 MHz, DMSO-d₆): δ1.87(s,3H); 2.88(dd, 1H); 3 .05 (dd, 1H);3.38 (d, 1H); 3.55-3.77(m, 2H) superimposed by 3.68(s,3H); 3.84 (d, 1H);4.24 (dd, 1H); 4.62(m, 1H); 4.90 (dd, 1H); 6.43(s, 1H); 6.70(d, 1H);7.16(d, 1H); 8.42(d, 1H); 9.27 (s,1H); 9.45(s,2H) ppm

EXAMPLE 152

Operating in an analogues manner as described in Example 150, butreplacing acetone by cyclopentanone, and using the product of Example112 (a) as starting material, there was obtained methyl (4R,7S)-7-(cyclopentylamino)-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylate.

¹ H-NMR(250 MHz, DMSO-d₆): δ1.15-1.80 (m, ˜9H); 1.88 (s, 3H); 2.87 (dd,1H); 2.98-3.18 (m, 2H); 3.35-3.45 (m, 2H); 3.65 (s, 3H); 3.77 (d, 1H);4.24 (dd, 1H); 4.54 (m, 1H); 4.85 (dd, 1H); 6.44 (s, 1H); 8.43 (d, H);9.49 (s, 1H); 9.51 (s, 1H) ppm

EXAMPLE 153

To a solution of 61 mg of the product of Example 14(a) in 0.6 mltetrahydrofuran/methanol (1:1, v/v), cooled to 0° C., were added 76 mgof sodium borohydride. The mixture was stirred for 1 hour at 0° C., thendiluted with ethyl acetate, and washed with saturated sodium carbonatesolution and with brine. The organic layer was dried over sodium sulfateand evaporated in vacuo. The residue was chromatographed on silica gelusing ethyl acetate as eluent to give 22 mg of12,14-bis(tert-butyldimethylsilylated)-product. This material wastreated with ammonium fluoride in methanol in an analogous manner to theprocedure described in Example 13 to yield, after crystallization frommethylene chloride/ethyl acetate/hexane, 10 mg of (4R,7S)-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-4-hydroxymethyl-7-(isopropylamino)-11-methyl-9,2,5-benzoxathiaazacyclododecine-6,10-dioneas a white solid.

¹ H-NMR (250 MHz, DMSO-d₆): δ(inter alia) 0.97 (d, 3H); 1 02 (d, 3H);1.88(s,3H); 2.72-2.90 (m, 2H); 3.28-3.45 (m, 4H); 3.76 (d, 1H); 3.89 (m,1H); 4.20 (dd, 1H); 4.82 (t,1H); 4.97(dd, 1H); 6.43(s, 1H); 7.81(d, 1H);9.44(s,1H); 9.47 (s, 1H) ppm

EXAMPLE 154

A solution of solution of 197 mg of tert-butyl (4R,7S)-12,14-bis-(tert-butyldimethylsilyloxy)-1,3,4,5,6,7,8,10-octahydro-4-hydroxymethyl-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamate,78 mg of p-chlorobenzoyl chloride, and 60 mg of 4-dimethylamino-pyridinewas stirred at 20° C. for 3 hours. The mixture was diluted with ethylacetate and washed successively with 3N hydrochloric acid, water, 5%sodium bicarbonate solution, and with brine. The organic layer was driedover sodium sulfate and evaporated in vacuo. The residue was dissolvedin 2 ml of trifluoroacetic acid, and the solution was stirred at 0° C.for 30 minutes. The solvent was evaporated in vacuo, and the residue wastreated with ammonium fluoride in methanol in an analogous manner to theprocedure described in Example 13 to yield, after chromatographicpurification on silica gel using ethyl acetate/hexane (1:1, v/v) aseluent, and crystallization from ethyl acetate/hexane, 28 mg of[(4R,7S)-7-amino-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecin-4-yl]methyl p-chlorobenzoate as a whitesolid.

¹ H-NMR(250 MHz, DMSO-d₆): δ1.87(s,3H); 2.63(dd, 1H); 2.95(dd, 1H);3.45(d, 1H); 3.61(m, 1H); 3.82 (d, 1H); 4.11(dd, 1H); 4.16-4.38(m,3H);5.17(m, 1H); 6.44(s, 1H); 7.61 (d, 2H); 7.98 (d, 2H); 8.13 (d, 1H); 9.46(s, 1H); 9.48 (s, 1H) ppm

EXAMPLE 155

Methyl (4R,7S)-14-(tert-butyldimethylsilyloxy)-1,3,4,5,6,7,8,10-octahydro-12-methoxy-11-methyl-7-dimethylamino-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylate was treated with ammonium fluoride in methanol in ananalogous manner to the procedure described in Example 13 to yield,after crystallization from ethyl acetate/hexane, 11 mg of methyl (4R,7S)-1,3,4,5,6,7,8,10-octahydro-14-hydroxy-12-methoxy-11-methyl-7-dimethylamino-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylateas a white solid.

¹ H-NMR(250 MHz, DMSO-d₆): δ1.92(s,3H); 2.31(s,6H); 2.83(dd, 1H);3.06(dd, 1H); 3.43(d, 1H); 3.64 (s, 3H); 3.72 (s, 3H); 3.85 (d, 1H);4.37 (dd, 1H); 4.55 (m, 1H); 4.82 (dd, 1H); 6.50 (s, 1H); 8.10 (d, 1H);9.75 (s, 1H) ppm

The starting material used above was prepared as follows:

(a) A solution of 100 mg of the product of Example 119(a) and 1.0 g ofmethyl iodide in 2 ml of acetonitrile was stirred at 20° C. for 3 hours.The solution was diluted with ethyl acetate and washed successively withsaturated sodium bicarbonate solution and with brine. The organic layerwas dried over sodium sulfate and evaporated in vacuo, and the residualoil was chromatographed on silica gel using ethyl acetate/hexane (2:1,v/v) as eluent to yield 17 mg of methyl (4R,7S)-14-(tert-butyldimethylsilyloxy)-1,3,4,5,6,7,8,10-octahydro-12-methoxy-11-methyl-7-dimethylamino-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylate,and 23 mg of methyl (4R,7S)-14-(tert-butyldimethylsilyloxy)-1,3,4,5,6,7,8,10-octahydro-12-methoxy-11-methyl-7-methylamino-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylate.

EXAMPLE 156

A solution of 45 mg of the product of Example 104 and 137 mg ofbromoacetone in 0.9 ml of ethanol was heated to 80° C. for 2 hours. Thesolution was diluted with ethyl acetate, washed successively with 5%sodium bicarbonate solution and with brine, dried over sodium sulfate,and evaporated in vacuo. The residue was chromatographed on silica gelusing ethyl acetate/hexane (1:2, v/v) as eluent, and the purifiedproduct was crystallized from ethyl acetate/hexane to give 6 mg oftertbutyl (4R,7S)-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-11-methyl-4-(4-methyl-thiazol-2-yl)-6,10-dioxo-9,2,5-benzoxathiaaza-cyclododecine-7-carbamateas a white solid.

¹ H-NMR(250 MHz,DMSO-d₆): δ1.42(s,9H); 1.90(s,3H); 2.33(s,3H); 2.92(dd,1H); 3.26 (m,partially obcsured, 1H); 3.56(d, 1H); 3.92 (d, 1H);4.25(dd, 1H); 4.37 (m, 1H); 4.97(dd, 1H); 5.25(m, 1H); 6.46(s, 1H);7.17(d, 1H); 7.21 (s, 1H); 8.48 (d, 1H); 9.50 (s, 1H); 9.53 (s, 1H) ppm

EXAMPLE 157

A mixture of 85 mg of(4R,7S)-12,14-bis-(tert-butyldimethylsilyloxy)-4-carbazoyl-4,5,6,7,8-tetrahydro-7-hydroxy-11-methyl-9,2,5-benzoxathiaazacyclododecine-6,10-dionein 5 ml of ethyl acetate and 5 ml of 1N hydrochloric acid, cooled to 0°C., was treated with 25 mg of sodium nitrite, and then stirred for 15minutes at 0° C. The phases were separated, and the organic phase waswashed with brine, dried over sodium sulfate, and evaporated in vacuo.The residue was taken up in 1 ml of dimethylformamide, 25 mg ofN-hydroxyacetamide were added, and the mixture was heated to 100° C. for1.5 hours. The solvent was evaporated in vacuo and the residue waschromatographed on silica gel using ethyl acetate/hexane (1:1, v/v) aseluent. The 12,14-bis-(tert-butyldimethylsilylated)-product obtained wastreated with ammonium fluoride in methanol in an analogous manner to theprocedure described in Example 13 to yield 16 mg of (4R,7S)-1,3,4,5,6,7,8,10-octahydro-7,12,14-trihydroxy-11-methyl-4-(3-methyl-1,2,4-oxadiazol-5-yl)-9,2,5-benzoxathiaazacyclododecine-6,10-dioneas a white solid, m.p. 265°-267° C.

The starting material used above was prepared as follows:

(a) A solution of 170 mg of the12,14-bis-(tert-butyldimethylsilylated)-product of Example 39 and 0.05ml of hydrazine hydrate in 2 ml of methanol was heated at reflux for 45minutes. The solution was evaporated in vacuo and the residue wascrystallized from ethyl acetate to give 95 mg of(4R,7S)-12,14-bis-(tert-butyldimethylsilyloxy)-4-carbazoyl-4,5,6,7,8-tetrahydro-7-hydroxy-11-methyl-9,2,5-benzoxathiaazacyclododecine-6,10-dioneas white needles of m.p. 242°-244° C.

EXAMPLE 158

A solution of 97 mg of the product of Example 2 and 0.2 ml of hydrazinehydrate in 1 ml of methanol was stirred at 20° C. for 45 minutes. Thesolution was diluted with ethyl acetate, and washed with 0.5M sodiumdihydrogenphosphate solution and with water. The organic layer was driedover sodium sulfate and evaporated in vacuo, and the residue wascrystallized from methanol/ethyl acetate/hexane to give 85 mg oftert-butyl (4R,7S)-4-carbazoyl-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamateas a white solid. as a white solid.

¹ H-NMR(250 MHz, DMSO-d₆): δ1.42 (s, 9H); 1.89 (s, 3H); 2.63 (dd, 1H):2.94 (dd, 1H); 3.44 (d, 1H); 3.86 (d, 1H); 4.15 (dd, 1H); 4.26 (broad s,2H); 4.47 (m, 1H); 4.92 (dd, 1H); 6.44 (s, 1H); 7.46 (d, 1H); 7.95 (d,1H); 9.10 (s, 1H); 9.48s, 1H) ppm

EXAMPLE 159

A solution of 37.5 mg of tert-butyl (4R,7S)-14-(tert-butyldimethylsilyloxy)-1,3,4,5,6,7,8,10-octahydro-4-methoxycarbonyl-6,10-dioxo-12-(1,3-dioxo-1,3-dihydro-isoindol-2-yl)9,2,5-benzoxathiaazacyclododecine-7-carbamateand 5.8 mg of 3-dimethylaminopropylamine in 1.5 ml of methanol wasstirred at 20° C. for 3 hours. Then, 30 mg of ammonium fluoride wereadded, and stirring was continued for another 2 hours. The solvent wasevaporated in vacuo, and the residue was partitioned between ethylacetate and brine. The organic layer was dried over sodium sulfate, thesolvent was evaporated in vacuo, and the residue was chromatographed onsilica gel, using methylene chloride/methanol (19: 1, v/v) as eluent, toyield, after crystallization from diethylether, 11 mg of tert-butyl (4R,7S)-12-amino-14-hydroxy-1,3,4,5,6,7,8,10-octahydro-4-methoxycarbonyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamateas a white solid.

¹ H-NMR (250MHZ,DMSO-d₆): δ1.42(s,9H); 2.82(dd, J=14 Hz and 10 Hz,1H);3.02(dd, J=14 Hz and 10 Hz, 1H); 3.64 (s, 3H); 3.80 (d, J=10 Hz, 1H);4.12-4.42 (m, 3H); 4.54-4.78(m,2H); 5.25(broad s,2H); 6.26(d,J=2 Hz,1H); 6.55(broad s, 1H); 7.36(d,J=8 Hz, 1H); 8.24(d,J=8 Hz, 1H); 9.39(s,1H) ppm

The starting material used above was prepared as follows:

(a) A mixture of 7.00 g of 3,5-dihydroxy-2-methylbenzoic acid, 5.40 g ofammonium chloride, and 20 ml of 25% aqueous ammonia was heated to 180°C. for 40 hours in a pressure vessel. After cooling, the mixture wasevaporated in vacuo. The residue was taken up in 230 ml of 6Nhydrochloric acid, and the mixture was heated at reflux for 16 hours.After cooling, unsoluble material was removed by filtration, and thefiltrate was evaporated in vacuo. The residue was supended in 100 ml ofwater, the pH of the suspension was adjusted to 4.5 by the addition of3N sodium hydroxide, and the mixture was cooled to 0° C. for 30 minutes.The precipitate was isolated by filtration and dried to give 2.50 g of5-amino-3-hydroxy-2-methylbenzoic acid as white crystals, m.p. >250° C.

¹ H-NMR(250 MHz,DMSO-d₆): δ2.11(s,3H); 6.24(d,J=2.5 Hz, 1H); 6.47(d,J=2.5 Hz, 1H); 9.08 (s, 1H) ppm

(b) A mixture of 300 mg 5-amino-3-hydroxy-2-methylbenzoic acid and 265mg of phtalic anhydride in 12 ml of dimethylformamide was heated to 110°C. for 2 hours, and subsequently to 150° C. for another 2 hours. Thesolvent was evaporated in vacuo and the residue was crystallized fromethanol/diethylether to give 310 mg of5-(1,3-dioxo-1,3-dihydro-isoindol-2-yl)-3-hydroxy-2-methylbenzoic acidas white crystals, m.p. >250° C.

¹ H-NMR (250 MHz,DMSO-d₆): δ2.36(S,3H); 7.07 (d,J=2.5 Hz, 1H);7.29(d,J=2.5 Hz, 1H); 7.80-8.00(4H); 10.06(S, 1H) ppm

(c) A mixture of 120 mg of5-(1,3-dioxo-1,3-dihydro-isoindol-2-yl)-3-hydroxy-2-methylbenzoic acid,87 mg of 4-nitrobenzyl bromide and 139 mg of potassium carbonate in 3 mlof dimethylformamide was stirred at 0° C. for 4 hours. The solvent wasevaporated in vacuo, and the residue was triturated with a mixture ofmethylene chloride and saturated sodium bicarbonate solution. Theunsoluble material was isolated by filtration to yield 190 mg ofp-nitrobenzyl5-(1,3-dioxo-1,3-dihydroisoindol-2-yl)-3-hydroxy-2-methylbenzoate, m.p.210°-244° C.

¹ H-NMR(250 MHz, DMSO-d₆): δ2.35(s,3H); 5.47(s,2H); 7.10(d,J=3 Hz, 1H);7.32(d,J=3 Hz,1H); 7.72 (d, J=9 Hz, 2H); 7.9-8.0 (m, 4H); 8.25 (d, J=9Hz, 2H) ppm

(d) To a suspension of 1.0 g of p-nitrobenzyl5-(1,3-dioxo-1,3-dihydro-isoindol-2-yl)-3-hydroxy-2-methylbenzoate in 30ml of dimethylformamide were added 0.71 g oftert-butyldimethylchlorosilane and 0.48 g of triethylamine, and themixture was stirred at 20° C. for 24 hours. The solvent was evaporatedin vacuo, and the residue was partitioned between 2N hydrochloric acidand ethyl acetate. The organic layer was washed with brine, dried oversodium sulfate, and the solvent was evaporated in vacuo. The residualoil was chromatographed on silica gel using methylene chloride as eluentto yield 0.90 g of p-nitrobenzyl3-(tert-butyldimethylsilyloxy)-5-(1,3-dioxo-1,3-dihydro-isoindol-2-yl)-2-methylbenzoate as white solid, m.p. 125°-127° C.

(e) p-Nitrobenzyl3-(tert-butyldimethylsilyloxy)-5-(1,3-dioxo-1,3-dihydro-isoindol-2-yl)-2-methylbenzoatewas subjected in an analogous manner to a sequence of proceduresdescribed in Examples 1 (d, f) and 13 (f), and the resulting product wassubjected in an analogous manner to the cyclization procedure describedin Example 21 to give tert-butyl(4R,7S)-14-(tertbutyldimethylsilyloxy)-1,3,4,5,6,7,8,10-octahydro-4-methoxycarbonyl-6,10-dioxo-12-(1,3-dioxo-1,3-dihydro-isoindol-2-yl)-9,2,5-benzoxathiaazacyclododecine-7-carbamateas a white solid.

EXAMPLE 160

A solution of 47 mg of the product of Example 159 and 27 mg of sodiumborohydride in 5 ml of tetrahydrofuran was added within 10 minutes to amixture of 0.08 ml of 6N sulfuric acid and 0.05 ml of 40% aqueousformaldehyde in 2 ml of tetrahydrofuran at 0° C. Stirring was continuedfor 4 hours at 0° C. The mixture was diluted with ethyl acetate, andthen washed with 10% sodium carbonate solution and with brine. Theorganic layer was dried over sodium sulfate and evaporated in vacuo. Theresidue was crystallized from ethyl acetate/diethylether to give 25 mgof tert-butyl (4R,7S)-1,3,4,5,6,7,8,10-octahydro-14-hydroxy-4-methoxycarbonyl-12-dimethylamino-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamateas a white solid.

¹ H-NMR(250MHZ,DMSO-d₆): δ1.42 (s, 9H); 2.60-2.85 (m, 1H) superimposedby 2.85 (s, 6H); 3.04(dd, J=14 Hz and 4 Hz, 1H); 3.64(s,3H); 3.86(d,J=10Hz, 1H); 4.12-4.93 (m, 3H); 4.14 (m, 1H); 4.80 (m 1H); 6.38 (d, J=1.5Hz, 1H); 6.63(broad s;1H); 7.40(d,J=7 Hz, 1H); 8.28(d,J=SHz, 1H); 9.61(s, 1H) ppm

EXAMPLE 161

A mixture of 25 mg of the product of Example 159 and 0.1 ml of pyridinein 1.5 ml of acetic anhydride was stirred at 20° C. for 3 hours. Theprecipitate was collected by filtration, washed with pentane, anddissolved in 1.5 ml of methanol. After the addition of 2.6 mg ofpotassium carbonate, the mixture was stirred for 1.5 hours at 20° C.,and then evaporated in vacuo. Water was added to the residue, and theinsoluble material was isolated by filtration, to yield 7 mg oftert-butyl(4R,7S)-12-acetylamino-1,3,4,5,6,7,8,10-octahydro-14-hydroxy-4-methoxycarbonyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamateas a white solid.

¹ H-NMR(250MHZ, DMSO-d₆): δ1.42 (s, 9H); 2.02 (S, 3H); 2.88 (dd, 1H);3.07 (dd, 1H); 3.64 (S, 3H); 3.88 (d, 1H); 4.24 (d, 1H); 4.28-4.44 (m,2H); 4.58-4.83 (m, 2H); 7.38 (d, 1H); 7.48 (d, 1H); 7.63 (d, 1H); 8.28(1H); 10.00 (s, 1H); 10.03 (s, 1H) ppm

EXAMPLE 162

A solution of 66 mg of the product of Example 119 and 0.15 ml ofpyridine in 1.5 ml of acetic anhydride was stirred at 20° C. for 3hours. The solution was evaporated in vacuo, the residue was dissolvedin ethyl acetate, and the solution was washed with 10% sodium carbonatesolution and with brine. The organic layer was dried over sodium sulfateand evaporated in vacuo. The residue was crystallized frommethanol/diethylether to give 33 mg of methyl(4R,7S)-7-acetylamino-14-acetoxy-1,3,4,5,6,7,8,10-octahydro-12-methoxy-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylateas a white solid.

¹ H-NMR(250 MHz, DMSO-d₆): δ1.93 (s, 3H); 2.05 (s, 3H); 2.91 (dd, 1H);3.15 (dd, 1H); 3.40 (d, 1H); 3.63 (s, 3H); 3.73 (d, 1H); 3.77 (s, 3H);4.13 (dd, 1H); 4.43 (dd, 1H); 4.69 (m, 1H); 5.09(dd, 1H); 6.88(s, 1H);8.29(d, 1H) ppm

EXAMPLE 163

A solution of 110 mg of crude tert-butyl (4R,7S)- and tert-butyl (4S,7S)-12,14-bis-(tert-butyldimethylsilyloxy)-1,3,4,5,6,7,8,10-octahydro-4-formyl-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamateand 73 mg of triphenylphosporanylidene-acetic acid tert-butylester in 1ml of toluene was stirred at 20° C. for 45 minutes. The solvent wasevaporated, and the residue was chromatographed on silica gel usingethyl acetate/hexane (1:1, v/v) as eluent. The majorbis-(tert-butyldimethylsilylated)-product obtained was treated withammonium fluoride in methanol in an analogous manner to the proceduredescribed in Example 13 to yield, after crystallization from ethylacetate/hexane, 17 mg of tert-butyl (E)-3-[(4R, 7S)- or tert-butyl(E)-3-[(4S,7S)-7-tert-butoxycarbonylamino-12,14-dihydroxy-11-methyl-6,10-dioxo-1,3,4,5,6,7,8,10-octahydro-9,2,5-benzoxathiaazacyclododecin-4-yl]-acrylate as a white solid.

¹ H-NMR(250 MHz, DMSO-d₆): δ1.41 (s, 9H); 1.42 (s, 9H); 1.87 (s, 3H);2.30 (dd, 1H); 2.95 (dd, 1H); 3.66 (d, 1H); 3.75 (d, 1H); 4.34-4.57 (m,3H); 4.62 (m, 1H); 5.99 (d, 1H); 6.43 (s, 1H); 6.73(dd, 1H); 7.24(d,1H); 8.52(d, 1H); 9.47(s,1H); 9.50 (s, 1H) ppm

(In addition, 6 mg of the 4S- or 4R-epimer were obtained).

The starting material used above was prepared as follows:

(a) Using in an analogous manner the procedure described in Example16(d), but replacing 3-trityloxy-1-propanol by the 12, 14-bis(tert-butyldimethylsilylated)-product of Example 58, there was obtainedcrude tert-butyl (4R,7S)-12,14-bis-(tertbutyldimethylsilyloxy)-1,3,4,5,6,7,8,10-octahydro-4-formyl-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamateas a mixture with its 4S-epimer.

EXAMPLE 164

To a solution of 156 mg of the product of Example 119(a) in 1 ml ofdimethylformamide were added 593 mg 4-bromo-phenacyl bromide and 219 mgof 4-dimethylamino-pyridine, and the mixture was heated to 40° C. for 4hours. The mixture was diluted with ethyl acetate and then washed with1N hydrochloric acid and with brine. The organic layer was dried oversodium sulfate and evaporated in vacuo. The residue was chromatographedon silica gel using ethyl acetate/hexane (2:1, v/v) as eluent. The14-(tert-butyldimethylsilylated)-Product obtained was treated withammonium fluoride in methanol in an analogous manner to the proceduredescribed in Example 13 to yield, after crystallization from ethylacetate/hexane, 36 mg of methyl (4R,7S)-7-[2-(4-bromo-phenyl)-2-oxo-ethylamino]-1,3,4,5,6,7,8,10-octahydro-14-hydroxy-12-methoxy-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylateas a white solid.

¹ H-NMR (250 MHz, DMSO-d₆): δ1.91(s,3H); 2.85(dd, 1H); 3.09(dd, 1H);3.48(d, 1H); 3.55(m, 1H); 3.63 (s, 3H); 3.73 (s, 3H); 3.83 (d, 1H); 4.17(dd, 1H); 4.35 (dd, 1H); 4.40 (dd, 1H); 4.62 (m, 1H); 5.04 (dd, 1H);6.51 (s, 1H); 7.74 (d, 2H); 7.87 (d, 2H); 8.43 (d, 1H); 9.73 (d, 1H) ppm

EXAMPLE 165

A solution of 103 mg of the product of Example 119(a) and 24 mg ofsuccinic anhydride in 2 ml of acetonitrile were heated to 70° C. for 1hour. The solution was cooled to 20° C., and 2 ml of methanol and 40 mgof ammonium fluoride were added. The mixture was stirred for 1 hour,then diluted with ethyl acetate and extracted with 5% sodium bicarbonatesolution and with water. The pH of the combined aqueous layer wasadjusted to 2 by the addition of 3N hydrochloric acid, and thenextracted with ethyl acetate. The ethyl acetate extract was washed withbrine, dried over sodium sulfate, and evaporated in vacuo. The residuewas crystallized from acetone/hexane to yield 59 mg of methyl (4R,7S)-7-(3-carboxy-propionylamino)-1,3,4,5,6,7,8,10-octahydro-14-hydroxy-12-methoxy-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylateas a white solid.

¹ H-NMR(250 MHz,DMSO-d₆): δ1.93(s,3H); 2.30-2.55(m, partially obscured,4H); 2 .86 (dd, 1H); 3.09 (dd, 1H); 3.52 (d, 1H); 3.62 (s, 3H); 3.72 (s,3H); 3.78 (d, 1H); 4.10 (dd, 1H); 4.46 (m, 1H); 4.64 (m, 1H); 5.10 (dd,1H); 6.52 (s, 1H); 8.20 (d, 1H); 8.35(d, 1H); 9.74(s,1H); 12.10(broad s,1H) ppm

EXAMPLE 166

The product of Example 110 was acylated withN-(tert-butoxycarbonyl)-β-alanine in an analogues manner to theprocedure described in Example 122. The resulting product was treatedwith trifluoroacetic acid at 0° C. for 30 minutes to yield, afterevaporation of the solvent and trituration of the residue withdiethylether, methyl (4R,7S)-7-(3-aminopropionylamino)-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylate trifluoroacetate as awhite solid.

¹ H-NMR(250 MHz,DMSO-d₆): δ2.50-3.20 (ca. 20H); 3.62 (s, 3H); 3.80-3.95(m, 3H); 4.14 (m, 1H); 4.34 (dd, 1H); 4.54 (m, 1H); 4.65 (m, 1H); 4.87(m, 1H); 6.52 (d, 1H); 6.72 (d, 1H); 7.78(broad s,2H); 8.49(m, 1H);8.67(m, 1H); 9.67(s,1H); 9.89 (s, 1H) ppm

EXAMPLE 167

A mixture of 31 mg of the product of Example 14 (a), 4.3 mg ofpivalaldehyde, and 12 mg of magnesium sulfate in 3 ml of methylenechloride was stirred for 5 hours at 20° C. The mixture was diluted withmethylene chloride, washed with water, and the organic layer was driedover sodium sulfate. The bis-(tert-butyldimethylsilylated)-productobtained was treated with ammonium fluoride in methanol in an analogousmanner to the procedure described in Example 13 to yield, aftercrystallization from diethylether, 8 mg of methyl (E or Z) (4R,7S)-7-(tert-butylmethylene-amino)-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylatetrifluoroacetate as a white solid.

¹ H-NMR(250 MHz,CDCl₃): δ1.16 (s, 9H); 2.01 (s, 3H); 2.77 (dd, 1H); 3.08(dd, 1H); 3.71 (d, 1H); 3.77 (s, 3H); 4.02 (m, 1H); 4.38 (d, 1H); 4.56(dd, 1H); 4.98 (dd, 1H); 5.15-5.25 (m, 2H); 6.43 (s, 1H); 7.16 (s, 1H);7.81 (s, 1H); 8.11 (d, 1H) ppm

EXAMPLE 168

A solution of 74 mg of trimethyloxonium tetrafluoroborate in 0.5 ml ofacetonitrile was stirred at 20° C. for 3 hours, and then added to asolution of 51 mg of the product of Example 119(a) in 0.1 ml ofacetonitrile, and stirring was continued for 2 hours. The solution wasdiluted with ethyl acetate, washed successively with saturated sodiumcarbonate solution and with brine, dried over sodium sulfate, andevaporated in vacuo. The residue was chromatographed on silanised silicagel using ethyl acetate/hexane (2:1, v/v) as eluent. The(tert-butyldimethylsilylated)-product obtained was treated with ammoniumfluoride in methanol in an analogous manner to the procedure describedin Example 13 to yield, after chromatographic purification on MCI-GelCHP20P using 0-70% aqueous acetonitrile as eluent and lyophilization ofthe product-containing fractions, 14 mg of (E)- or (Z)-(4R,7S)-7(1-methylimino-ethylamino)-1,3,4,5,6,7,8,10-octahydro-14-hydroxy-12-methoxy-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylicacid methylester as a white solid.

¹ H-NMR(250 MHz, DMSO-d₆): (inter alia) δ1.88(s,3H); 2.83(dd, 1H);2.99(dd, 1H); 3.53(d, 1H); 3.65 (s, 3H); 3.71 (s, 3H); 4.25 (d, 1H);4.38 (dd, 1H); 4.70 (m, 2H); 4.83 (dd, 1H); 6.52 (s, 1H); 8.43 (d, 1H)ppm

Mass spectrum: m/z 454 (M+H)

EXAMPLE 169

To a solution of 90 mg of(4R,7S)-7-tert-butoxycarbonylamino-14-tert-butyldimethylsilyloxy-1,3,4,5,6,7,8,10-octahydro-12-methoxy-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylicacid in 1.5 ml of tetrahydrofuran were added 49 mg ofdipyridyl-2,2-disulfide and 79 mg of triphenylphosphine. The solutionwas stirred at 0° C. for 1 hour, and then 0.15 ml of a 1M solution ofethylmagnesium bromide in tetrahydrofuran were added. The mixture wasstirred at 0° C. for 30 minutes, and then 0.5 ml of water were added,and stirring was continued for 5 minutes. The mixture was diluted withethyl acetate and washed successively with 1N hydrochloric acid and withbrine. The organic phase was dried over sodium sulfate, evaporated invacuo, and the residual oil was chromatographed on silica gel usingacetone/hexane (1:4, v/v) as eluent. The(tert-butyldimethylsilylated)-product obtained was treated with ammoniumfluoride in methanol in an analogous manner to the procedure describedin Example 13 to yield 5 mg of tert-butyl (4R,7S)-1,3,4,5,6,7,8,10-octahydro-14-hydroxy-12-methoxy-11-methyl-4-propionyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamateas pale yellow foam.

¹ H-NMR(400 MHz,CDCl₃): (inter alia) δ1.06(t,3H); 1.45(s,9H);2.06(s,3H); 2.53(m, 2H); 2.90-3.20(m, 2H); 3.49(d, 1H); 3.77(s,3H);3.93(d, 1H); 6.50(s, 1H) ppm

Mass spectrum: m/z (inter alia) 519 (M+Na); 497 (M+H); 441 (M+H-C₄ H₈)

The starting material used above was prepared as follows:

(a) A mixture of 0.48 g of the product of Example 51, 0.45 g oftert-butyldimethylchlorosilane, and 0.40 g of triethylamine in 2 ml ofdimethylaminoformamide was heated with stirring to 40° C. for 2 hours.The mixture was partitioned between ethyl acetate/hexane (1:1, v/v) andice-cold 5% aqueous sodium bicarbonate solution. The organic phase waswashed with 10% sodium chloride solution, dried over sodium sulfate, andevaporated in vacuo. The residual oil was dissolved in 3 ml of methanol,and the solution was stirred at 40° C. for 2 hours. The solvent wasevaporated in vacuo, and the residue was stirred for 1 hour with 10 mlof hexane at 0° C. to give 0.54 g of (4R,75)-7-(tert-butoxycarbonylamino)-14-(tert-butyldimethylsilyloxy)-1,3,4,5,6,7,8,10-octahydro-12-methoxy-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylicacid as white crystals.

EXAMPLE 170

To a solution of 130 mg of the12,14-bis-(tert-butyldimethylsilylated)-product of Example 120 in 4 mlof methylene chloride, cooled 0° C., were added 63 mg of 55%3-chloroperbenzoic acid. The solution was stirred at 0° C. for 30minutes, then diluted with methylene chloride, washed successively withsaturated sodium bicarbonate solution and with brine, dried over sodiumsulfate, and evaporated in vacuo. The residual oil was chromatographedon silica gel using acetone/hexane (1:5, v/v) as eluent to afford twobis-(tert-butyldimethylsilylated)-products. The product eluated firstwas treated with ammonium fluoride in methanol in an analogous manner tothe procedure described in Example 13 to yield 34 mg of methyl (2R, 4R,7S)- or methyl (2S, 4R,7S)-7-acetylamino-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylate2-oxide as a white solid.

¹ H-NMR(250 MHz, DMSO-d₆): δ1.90(s,3H); 1.94(s,3H); 3.01(dd, 1H);3.64(s,3H); 3.77(dd, 1H); 3.85 (d, 1H); 4.01 (dd, 1H); 4.18 (d, 1H);4.62 (m, 1H); 4.98 (m, 1H); 5.09 (dd, 1H); 6.52 (s, 1H); 8.26(d, 1H);8.79(broad s, 1H); 9.63(s, 1H); 9.73 (s, 1H) ppm

EXAMPLE 171

To a solution of 48,5 mg of the product of Example 2 in 0.5 ml ofmethanol were added 10.5 mg of hydroxylamine hydrochloride and 0.5 ml ofa 0.3N solution of potassium hydroxyde in methanol. The solution wasstirred for 20 hours at 20° C., then diluted with ethyl acetate andwashed with water. The organic layer was dried over sodium sulfate andevaporated in vacuo. The residue was chromatographed on silica gel usingethyl acetate as eluent to yield, after crystallization from ethylacetate/hexane 12 mg of tert-butyl (4R,7S)-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-4-hydroxycarbamoyl-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-7-carbamateas a white solid.

¹ H-NMR(250 MHz, DMSO-d₆): δ1.42(S,9H); 1,89(S,3H); 2.65(dd, 1H);2.91(dd, 1H); 3.45(d, 1H); 3.87 (d, 1H); 4.15 (dd, 1H); 4.31 (m, 1H),4.42 (m, 1H); 4.92 (dd, 1H); 6.46 (s, 1H); 7.44 (d, 1H), 7.95 (d, 1H);8.95 (broad s, 1H); 9.53 (s, 1H); 10.68 (broad s, 1H) ppm

EXAMPLE 172

To a solution of 90 mg of(R)-2-[[(R)-2-(2-amino-2-methoxycarbonyl-ethyl)thio]methyl]-3,5-bis(tert-butyldimethylsilyloxy)-6-methyl-benzoic acidin 3 ml of acetonitrile, cooled to 0° C., were added 38 mg ofN-(dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride. The mixturewas stirred at 0° C. for 1 hour, then diluted with 40 ml of ethylacetate and washed with 1N hydrochloric acid and with brine. The organiclayer was dried over sodium sulfate and evaporated in vacuo. The residuewas chromatographed on silica gel using ethyl acetate/hexane (1:1, v/v)as eluent to yield 36 mg of methyl (4R, 8R)-12,14-bis(tert-butyldimethylsilyloxy)-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-8,11-dimethyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylate.This material was treated with ammonium fluoride in methanol in ananalogous manner to the procedure described in Example 21 to yield,after crystallization from ethyl acetate/hexane, methyl (4R,8R)-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-8,11-dimethyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecine-4-carboxylateas a white solid.

¹ H-NMR(250 MHz, DMSO-d₆): δ1.31 (d, 3H); 1.68 (s, 3H); 2.37-2.76 (m,3H, partially obscured); 3.08 (dd, 1H); 3.32 (d, 1H); 3.33 (s, 3H); 3.96(d, 1H); 4.68 (m, 1H); 5.67 (m, 1H); 6.41 (s, 1H); 8.41 (d, 1H); 9.45(s, 1H); 9.48 (s, 1H) ppm

The starting material used above was prepared as follows:

(a) To a suspension of 5.5 g of L-cysteine methyl ester hydrochloride in80 ml of dioxane were added 3.6 g of 4-methylmorpholine and 7.0 g ofdi-tert-butyl-dicarbonate. After being stirred at 20° C. for 15 hours,the mixture was poured into ice-cold 0.2N hydrochorid acid, and theproduct was extracted with ethyl acetate. The organic layer was washedwith brine, dried over sodium sulfate, and the solvent was evaporated invacuo. The residual oil was chromatographed on silica gel using ethylacetate/hexane (1:4, v/v) as eluent to yield 2.87 g ofN-(tert-butoxycarbonyl-L-cysteine methyl ester as an oil.

(b) A solution of 5.0 g of p-Nitrobenzyl2-bromomethyl-3-(tert-butyldimethylsilyloxy)-5-methoxybenzoate and 1.88g of N-(tert-butoxycarbonyl-L-cysteine methyl ester in 40 ml ofmethylene chloride, cooled to 0° C., was treated with 0.81 oftriethylamine. The mixture was stirred for 30 minutes at 0° C. and for 1hour at room temperature, then diluted with ethyl acetate and washedwith 0.5N hydrochloric acid and with brine. The organic layer was driedover sodium sulfate, and the solvent was evaporated in vacuo. Theresidual oil was chromatographed on silica gel using ethylacetate/methylene chloride/hexane (1:2:4, v/v/v) as eluent to yield 3.64g of p-nitrobenzyl(R)-2-[[[(2-tert-butoxycarbonylamino-2-methoxycarbonyl)ethyl]thio]methyl]-3,5-bis(tert-butyldimethylsilyloxy)-6-methyl-benzoate as an oil.

(c) The product of the previous experiment was subjected in an analogousmanner to the procedure described in Example 13 (f) to yield(R)-2-[[[(2-tert-butoxycarbonylamino-2-methoxycarbonyl)ethyl]thio]methyl]-3,5-bis(tert-butyldimethylsilyloxy)-6-methyl-benzoic acid.

(d) A solution of 1.05 g of the product of the previous experiment, 0.52g of tert-butyl (S)-3-hydroxybutyrate, and 0.85 g of triphenylphosphinein 40 ml of toluene was cooled to 0° C. Then, 0.57 g of diethylazodicarboxylate were added, and the mixture was stirred for 15 minutesat 0° C. and for 2 hours at room temperature. The solvent was evaporatedin vacuo. The residue was stirred with diethylether/hexane 0° C. for 15minutes, unsoluble material was filtered off, and the filtrate wasevaporated in vacuo. The residue was chromatographed on silica gel usingethyl acetate/hexane (1:4, v/v) as eluent to give 0.92 g of(R)-2-[[[(2-tert-butoxycarbonylamino-2-methoxycarbonyl)ethyl]thio]methyl]-3,5-bis (tert-butyldimethylsilyloxy)-6-methyl-benzoicacid (R)-2-tert-butoxycarbonyl-1-methyl-ethyl ester as an oil.

(e) A solution of 0.79 g of the product of the previous experiment in 4ml of trifluoroacetic acid was stirred at 20° C. for 30 minutes. Thesolution was evaporated in vacuo, and the residue was chromatographed onsilica gel using ethyl acetate/hexane (1:2, v/v) as eluent, to give 0.32g of (R)-2-[[(R)-2-(2-amino-2-methoxycarbonyl-ethyl)thio]methyl]-3,5-bis (tert-butyldimethylsilyloxy)-6-methyl-benzoic acidas an oil.

EXAMPLE A Manufacture of Dry Ampoules for Intramuscular Administration

A lyophilisate of 1 g of t-butyl(4R,7S)-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-4-methoxycarbonyl-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazabicyclododecine-7-carbamateis prepared in the usual manner and filled into an ampoule. Prior to theadministration, the lyophilisate is treated with 2.5 ml of a 2% aqueouslidocaine hydrochloride solution.

EXAMPLE B

Interlocking gelatin capsules each containing the following ingredientsare manufactured in the usual manner:

    ______________________________________                                        t-butyl (4R,7S)-1,3,4,5,6,7,8,10-octahydro-12,14-                                                       500     mg                                          dihydroxy-4-methoxycarbonyl-11-methyl-6,10-                                   dioxo-9,2,5-benzoxathiaazacyclododecine-                                      7-carbamate                                                                   Luviskol (water-soluble polyvinylpyrrolidone)                                                           20      mg                                          Mannitol                  20      mg                                          Talc                      15      mg                                          Magnesium stearate        2       mg                                                                    557     mg                                          ______________________________________                                    

We claim:
 1. Process for the manufacture ofN-[[(4R,7S)-1,3,4,5,6,7,8,10-octahydro-12,14-dihydroxy-7-[[(3R)-3-hydroxy-1-L-seryl-L-prolyl]amino]-11-methyl-6,10-dioxo-9,2,5-benzoxathiaazacyclododecin-4-yl]carbonyl]-L-alanineand of pharmaceutically acceptable salts thereof, which comprisescultivating a subculture of the microorganism Streptomyces sp. NR0484FERM-BP-1982 under submerged, aerobic conditions in an aqueouscarbohydrate solution containing nitrogenous nutrients and sodiumchoride, thereafter isolating the desired compound from the fermentationbroth and, if desired. converting the product obtained into apharmaceutically acceptable salt thereof.
 2. A biologically pure cultureof Streptomyces sp. NR0484 FERM-BP-1982.